Synthetic mimetics of host defense and uses thereof

ABSTRACT

The present invention provides arylamide compounds and methods of making and using them as antibiotics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 61/108,595 filed Oct. 27, 2008, which is incorporated herein byreference in its entirety.

REFERENCE TO GOVERNMENT GRANTS

The present invention was supported by funds from the U.S. Government(NIH Grant Nos. AI74866 and 1R43AI058407) and the U.S. Government maytherefore have certain rights in the invention.

FIELD OF THE INVENTION

The present invention is directed, in part, to arylamide compounds andto methods of making and using the same.

BACKGROUND OF THE INVENTION

Antimicrobial peptides (AMPs) represent a first line of defense againstmicrobes for many species. AMPs are typically small (12-80 amino acids)cationic amphiphiles. There are two types of AMPs comprising ribosomallyand nonribosomally synthesized peptides. Over 700 AMPs have beenidentified and are generally α-helical (magainin and cecropin) ordisulfide-rich β-sheets (bactenecin and defensin). Although the peptidesare composed of many different sequences, their physiochemicalproperties are remarkably similar. They adopt an amphiphilicarchitecture with positively charged groups segregated to one side ofthe secondary structure and hydrophobic groups on the opposite surface.In mammals, the peptides are produced and secreted in skin, mucosalsurfaces and neutrophils, and act locally in response to infection. Itis the overall physiochemical properties that are largely responsiblefor biological activity of these peptides.

Some antimicrobial activities of host defense proteins have been linkedto direct cytotoxic actions and modulation of the innate immune system.Their direct antimicrobial activities are proposed to involve bothmembrane and non-membrane effects. Antimicrobial peptides have remainedan effective weapon against bacterial infection over evolutionary timeindicating that their mechanism of action thwarts bacterial responseswhich lead to resistance against toxic substances. This premise issupported by direct experimental data showing that no appreciableresistance to the action of the antimicrobial peptides occurs aftermultiple serial passages of bacteria in the presence of sub-lethalconcentrations of the peptides.

There is a dire need for development of new antimicrobial agents thatattack new targets to evade resistance issues that limit the usefulnessof many antibiotics. Furthermore, these new agents should exert theirantimicrobial activity via mechanisms that bacteria do not effectivelyresist. A series of non-peptidic analogues have been developed that havemany advantages over peptides because of their small size, whichincreases stability and enhances tissue distribution, and ability tofine-tune their physical properties for optimization of potency andsafety. A series of arylamide compounds that mimic structural propertiesof the antimicrobial peptides were found to have potent antibacterialactivities and wide selectivity ratios versus mammalian cells.

SUMMARY OF THE INVENTION

The present invention provides compounds of Formula I

wherein: each A is, independently, —C═O, —C═S, or —CH₂; each D is,independently, O or S; each R¹ is, independently, hydrogen, C₁₋₃alkyl,C₁₋₃alkoxy, halo, or haloC₁₋₃alkyl; each R² is, independently, hydrogen,C₁₋₃alkyl, C₁₋₃alkoxy, halo, or haloC₁₋₃alkyl; each R³ is,independently, hydrogen, C₁₋₄alkyl, C₁₋₄alkoxy, halo, or haloC₁₋₄alkyl;and each R⁴ is, independently, hydrogen, C₁₋₃alkyl, C₁₋₃alkoxy, halo, orhaloC₁₋₃alkyl; or a pharmaceutically acceptable salt thereof.

In some embodiments, each A is —C═O.

In some embodiments, each D is O.

In some embodiments, each R¹ is, independently, hydrogen, methyl, ethyl,methoxy, ethoxy, halo, or haloC₁₋₃alkyl. In some embodiments, each R¹is, independently, hydrogen, methyl, methoxy, halo, or haloC₁₋₃alkyl. Insome embodiments, each R¹ is, independently, hydrogen, methyl, ormethoxy. In some embodiments, each R¹ is hydrogen.

In some embodiments, each R² is, independently, hydrogen, methyl, ethyl,methoxy, ethoxy, halo, or haloC₁₋₃alkyl. In some embodiments, each R²is, independently, hydrogen, methyl, methoxy, or halo. In someembodiments, each R² is hydrogen.

In some embodiments, each R³ is, independently, hydrogen, methyl, ethyl,methoxy, ethoxy, halo, or haloC₁₋₃alkyl. In some embodiments, each R³is, independently, methyl, methoxy, halo, or haloC₁₋₃alkyl. In someembodiments, each R³ is, independently, halo or haloC₁₋₃alkyl. In someembodiments, each R³ is, independently, haloC₁₋₃alkyl. In someembodiments, each R³ is trifluoromethyl.

In some embodiments, each R⁴ is, independently, hydrogen, methyl, ethyl,methoxy, ethoxy, or haloC₁₋₃alkyl. In some embodiments, each R⁴ is,independently, hydrogen, methyl, methoxy, halo, or haloC₁₋₃alkyl. Insome embodiments, each R⁴ is, independently, hydrogen, methyl, methoxy,or halo. In some embodiments, each R⁴ is hydrogen.

In some embodiments, each A is, independently, —C═O, —C═S, or CH₂; eachD is, independently, O or S; each R¹ is, independently, hydrogen,methyl, ethyl, methoxy, ethoxy, halo, halomethyl, or haloethyl; each R²is, independently, hydrogen, methyl, methoxy, halo, or halomethyl; eachR³ is, independently, C₁₋₃alkyl, C₁₋₃alkoxy, halo, or haloalkyl; andeach R⁴ is, independently, hydrogen, methyl, ethyl, methoxy, ethoxy,halo, halomethyl, or haloethyl.

In some embodiments, each A is, independently, —C═O or —C═S; each D is,independently, O or S; each R¹ is, independently, hydrogen, methyl,methoxy, halo, or halomethyl; each R² is, independently, hydrogen, halo,or halomethyl; each R³ is, independently, methyl, ethyl, methoxy,ethoxy, halo, halomethyl, or haloethyl; and each R⁴ is, independently,hydrogen, methyl, ethyl, methoxy, ethoxy, halo, halomethyl, orhaloethyl.

In some embodiments, each A is —C═O; each D is O; each R¹ is,independently, hydrogen, halo, or halomethyl; each R² is, independently,hydrogen or halo; each R³ is, independently, methyl, methoxy, halo, orhalomethyl; and each R⁴ is, independently, hydrogen, methyl, methoxy,halo, or halomethyl.

In some embodiments, each A is —C═O; each D is O; each R¹ is,independently, hydrogen or halo; each R² is, independently, hydrogen orhalo; each R³ is, independently, methyl, halo, or halomethyl; and eachR⁴ is, independently, hydrogen, methyl, halo, or halomethyl.

In some embodiments, each A is —C═O; each D is O; each R¹ is,independently, hydrogen or halo; each R² is, independently, hydrogen orhalo; each R³ is, independently, halo or halomethyl; and each R⁴ is,independently, hydrogen or halo.

In some embodiments, each A is —C═O; each D is O; each R¹ is,independently, hydrogen or halo; each R² is, independently, hydrogen orhalo; each R³ is, independently, methyl, halo, or halomethyl; and eachR⁴ is, independently, hydrogen, methyl, halo, or halomethyl.

In some embodiments, the compound is

or a pharmaceutically acceptable salt thereof.

The present invention also provides pharmaceutical compositionscomprising one or more of the compounds described above or salt of anyof the compounds described above and a pharmaceutically acceptablecarrier.

The present invention also provides formulations comprising one or moreof the compounds described above, wherein the formulation comprisessaline, water, a cyclodextrin solution, or a buffered solution of pH 3to 9. In some embodiments, the formulation is an oral non-absorbedformulation. In some embodiments, the formulation comprises an excipientchosen from purified water, propylene glycol, polyethyleneglycol 400(PEG 400), glycerin, DMA, ethanol, benzyl alcohol, citric acid/sodiumcitrate (pH3), citric acid/sodium citrate (pH5),tris(hydroxymethyl)amino methane HCl (pH7.0), 0.9% saline, and 1.2%saline, or any combination thereof. In some embodiments, the formulationcomprises an excipient chosen from propylene glycol, purified water, andglycerin. In some embodiments, the formulation comprises an excipientchosen from 20% w/v propylene glycol in saline, 30% w/v propylene glycolin saline, 40% w/v propylene glycol in saline, 50% w/v propylene glycolin saline, 15% w/v propylene glycol in purified water, 30% w/v propyleneglycol in purified water, 50% w/v propylene glycol in purified water,30% w/v propylene glycol and 5 w/v ethanol in purified water, 15% w/vglycerin in purified water, 30% w/v glycerin in purified water, 50% w/vglycerin in purified water, 20% w/v Kleptose in purified water, 40% w/vKleptose in purified water, and 25% w/v Captisol in purified water.

The present invention also provides methods of preparing Compound Acomprising:

a) reacting (R)-(−)-N-Boc-3-pyrrolidinol with a strong base to form amixture; further reacting the mixture with2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene to form a compoundhaving Formula II

b) reacting the compound of Formula II with an alcohol and a transitionmetal catalyst in the presence of hydrogen to form a compound of FormulaIII

c) adding the compound of Formula III and pyrimidine-4,6-dicarboxylicacid to a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine andN-methylmorpholine to form a compound of Formula IV

d) reacting the compound of Formula IV with N-Boc-guanidine butyric acidto form a compound of Formula V

e) deprotecting the compound of Formula V to produce Compound A. In someembodiments, in a) the strong base is NaH; and in b) the transitionmetal catalyst is Pd/C and the alcohol is ethanol.

The present invention also provides alternate methods of preparingCompound A comprising:

a) deprotonating (R)-3-Hydroxypyrrolidine-1-carboxylic acid tent-butylester, and reacting the resultant compound with2-chloro-1,3-dinitro-5-trifluoromethylbenzene to form(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester;

b) reducing(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester in the presence of an alcohol, a transition metalcatalyst, and hydrogen to form(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester;

c) coupling(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester with pyrimidine-4,6-dicarboxylic acid in thepresence of 1-[(3-(dimethylamino)-propyl)]-3-ethylcarbodiimidehydrochloride to form pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide};

d) reacting pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}with({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)pentanoicacid in the presence of phosphorous oxychloride to formpyrimidine-4,6-dicarboxylic acidbis-{[3-(5-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-pentanoylamino)-2-((R)-1-(tert-butoxycarbonyl)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide};

e) deprotecting pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-pentanoylamino)-2-((R)-1-(tert-butoxycarbonyl)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}to form crude pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-guanidino-pentanoylamino)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}tetrahydrochloride;and

f) purifying crude pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-guanidino-pentanoylamino)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}tetrahydrochlorideby, for example, reverse-phase chromatography.

The present invention also provides second alternate methods ofpreparing Compound A comprising:

a) deprotonating (R)-3-Hydroxypyrrolidine-1-carboxylic acid tent-butylester and further reacting the resultant compound with2-chloro-1,3-dinitro-5-trifluoromethylbenzene to form(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester;

b) reducing(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester in the presence of an alcohol, a transition metalcatalyst, and hydrogen, to form(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester;

c) coupling(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester with pyrimidine-4,6-dicarboxylic acid in thepresence of 1-[(3-(dimethylamino)-propyl)]-3-ethylcarbodiimidehydrochloride to form pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide};

d) reacting pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}with N-Cbz acid in the presence of thionyl chloride;

e) reducing the resultant compound of d) in the presence of an alcohol,a transition metal catalyst, and hydrogen;

f) reacting the resultant compound of e) with di-Boc pyrazole; and

g) deprotecting the resultant compound off) to produce Compound A.

The present invention also provides methods of preparing apharmaceutically acceptable salt of Compound A comprising:

a) reacting (R)-(−)-N-Boc-3-pyrrolidinol with a strong base to form amixture; further reacting the mixture with2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene to form a compoundhaving Formula II

b) reacting the compound of Formula II with an alcohol and a transitionmetal catalyst in the presence of hydrogen to form a compound of FormulaIII

c1) adding the compound of Formula III and pyrimidine-4,6-dicarboxylicacid to a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine andN-methylmorpholine to form a compound of Formula IV

c2) adding the compound of Formula III and pyrimidine-4,6-dicarboxylicacid to a mixture of 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimidehydrochloride (EDCl) and anhydrous pyridine to form a compound ofFormula IV

d) adding the compound of Formula IV with an N-Cbz acid to a solutioncomprising anhydrous pyridine, dimethylaminopropylamine, and any one ofthionyl chloride, POCl₃, (EtO)₂POCl, or oxalyl chloride to form acompound of Formula Va

e) hydrogenlysis of the Cbz group of the compound of Formula Va toproduce the compound of formula VI

f) protecting the compound of Formula VI to produce the compound offormula VII

g) deprotecting the compound of Formula VII to produce apharmaceutically acceptable salt of Compound A.

The present invention also provides methods of inhibiting the growth ofa microbe comprising contacting the microbe with any of the compoundsdescribed above, or pharmaceutically acceptable salts thereof.

The present invention also provides methods of treating a mammal havinga microbial infection comprising administering to the mammal in needthereof an anti-microbial effective amount of any of the compoundsdescribed above, or pharmaceutically acceptable salts thereof.

In some embodiments, the microbe or microbial infection is agram-negative aerobe, a gram-positive aerobe, a gram-negative anaerobe,a gram-positive anaerobe, a mycobacterium or a yeast. In someembodiments, the gram-negative aerobe is Escherichia coli, Citrobacterfreundii, Citrobacter diverus, Citrobacter koseri, Enterobacter cloacae,Enterobacter faecalis, Klebsiella pneumonia, Klebsiella oxytoca,Morganella morganii, Providencia stuartii, Proteus vulgaris, Proteusmirabilis, Serratia marcescens, Acinetobacter haemolyticus,Acinetobacter junii, Acinetobacter lwoffii, Haemophilus influenzae,Stenotrophomonas maltophilia, or Pseudomonas aeruginosa. In someembodiments, the gram-positive aerobe is Enterococcus faecalis,Enterococcus faecium, Mycobacterium tuberculosis, Staphylococcus aureus,Staphylococcus pneumoniae, Staphylococcus epidermidis, Staphylococcussaprophyticus, Staphylococcus colmii, Staphylococcus sciuri,Staphylococcus warneri, Streptococcus agalactiae, Streptococcuspyogenes, Streptococcus anginosus, Streptococcus mitis, or Streptococcusoxalis. In some embodiments, the gram-negative anaerobe is Bacteroidesfragilis. In some embodiments, the gram-positive anaerobe is Clostridiumdifficile or Clostridium perfringens. In some embodiments, themycobacterium is Mycobacterium tuberculosis, Mycobacterium bovis,Mycobacterium africanum, Mycobacterium canetti, or Mycobacteriummicroti. In some embodiments, the yeast is Candida albicans or Candidakrusei.

The present invention also provides any of the compounds described abovefor treating a microbial infection.

The present invention also provides any of the compounds describedabove, or pharmaceutically acceptable salts thereof, for use in themanufacture of a medicament for the treatment of a microbial infection.

The present invention also provides use of any of the compoundsdescribed above, or pharmaceutically acceptable salts thereof, forinhibiting growth of a microbe.

The present invention also provides use of any of the compoundsdescribed above, or pharmaceutically acceptable salts thereof, fortreatment of a microbial infection in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B show time-kill studies of Compound A versus S.aureus ATCC27660 (FIG. 1B is expanded view of FIG. 1A).

FIG. 2 shows association of passage of S. aureus with norfloxacin with asignificant rise in MIC values by passage 3 (4 doubling dilutions) forboth MSSA and MRSA.

FIG. 3 shows efficacy of Compound A against S. aureus in the Mouse ThighBurden Model.

FIG. 4 shows efficacy of Compound A versus vancomycin against S. aureusin the Rat Thigh Burden Model.

FIG. 5 shows efficacy of Compound A against S. aureus in the MouseSepsis Model.

DESCRIPTION OF EMBODIMENTS

As used herein, the term “about” means±5% of the value it describes. Forexample, about 100 means from 95 to 105.

As used herein, the terms “C₁₋₃alkyl” “C₁₋₄alkyl” and “(CH₂)₁₋₇” meansaturated, monovalent unbranched or branched hydrocarbon chains havingfrom 1 to 3 carbons, from 1 to 4 carbons, and from 1 to 7 carbons,respectively. Examples of alkyl groups include, but are not limited to,(C₁-C₇)alkyl groups, such as methyl, ethyl, propyl, isopropyl,2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl,3-methyl-1-butyl, 2-methyl-3-butyl, 2-methyl-1-pentyl,2,2-dimethyl-1-propyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl,2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl,isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, and heptyl. Analkyl group can be unsubstituted or substituted with one or two suitablesubstituents.

As used herein, the terms “C₁₋₃alkoxy” and “C₁₋₄alkoxy” means —O-alkyl,with alkyl defined as above. An alkoxy group can be unsubstituted orsubstituted with one or two suitable substituents. The alkyl chain of analkyloxy group is from 1 to 3 or 1 to 4 carbon atoms in length.

As used herein, the term “halo” means a halogen such as fluorine,chlorine, bromine, or iodine.

As used herein, the terms “haloC₁₋₃alkyl” and “haloC₁₋₄alkyl” meansalkyl groups as defined above, wherein one or more (i.e., 1, 2, 3, 4, 5,6, 7, 8, 9, or 10) hydrogens are replaced with a halo, as defined above.

As used herein, “isolated” means that the compounds of Formula I areseparated from other components of either (a) a natural source, such asa cell, such as a bacterial culture, or (b) a synthetic organic chemicalreaction mixture, such as by conventional techniques, the compounds ofFormula I are purified.

As used herein, the term “mammal” means a rodent (i.e., a mouse, a rat,or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or ahuman. In some embodiments, the mammal is a human.

As used herein, the term “microbe” means a bacteria, fungi, protozoa, orvirus.

As used herein, the phrase “pharmaceutically acceptable salt(s)”includes, but is not limited to, salts of acidic or basic groups.

As used herein, the term “purified” means that, when isolated, theisolate contains at least 90%, at least 95%, at least 98%, or at least99% of a compound of Formula I by weight of the isolate.

As used herein, the phrase “suitable substituent” means a group thatdoes not nullify the synthetic or pharmaceutical utility of thecompounds of Formula I or the intermediates useful for preparing them.Examples of suitable substituents include, but are not limited to:(C₁-C₄)alkyl, (C₁-C₄)alkenyl, (C₁-C₄)alkynyl, (C₁-C₄)alkoxy, —CN, —OH,oxo, halo, —NO₂, —CO₂H, —NH₂, —NH((C₁-C₄)alkyl), —N((C₁-C₄)alkyl)₂,—CHO, —CO((C₁-C₄)alkyl), and —CO₂((C₁-C₄)alkyl). One of skill in art canreadily choose a suitable substituent based on the stability andpharmacological and synthetic activity of the compound of Formula I.

As used herein, the phrase “anti-microbial effective amount” of acompound comprising Formula I is measured by the anti-microbialeffectiveness of the compound. In some embodiments, an anti-microbialeffective amount inhibits growth of a particular microbe by at least10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%,by at least 60%, by at least 70%, by at least 80%, by at least 90%, orby at least 95%. In some embodiments, an “anti-microbial effectiveamount” is also a “therapeutically effective amount” whereby thecompound reduces or eliminates at least one harmful effect of a microbeon a mammal.

The present invention provides compounds of Formula I

wherein:

each A is, independently, —C═O, —C═S, or CH₂;

each D is, independently, O or S;

each R¹ is, independently, hydrogen, C₁₋₃alkyl, C₁₋₃alkoxy, halo, orhaloC₁₋₃alkyl;

each R² is, independently, hydrogen, C₁₋₃alkyl, C₁₋₃alkoxy, halo, orhaloC₁₋₃alkyl;

each R³ is, independently, hydrogen, C₁₋₄alkyl, C₁₋₄alkoxy, halo, orhaloC₁₋₄alkyl; and

each R⁴ is, independently, hydrogen, C₁₋₃alkyl, C₁₋₃alkoxy, halo, orhaloC₁₋₃alkyl;

or a pharmaceutically acceptable salt thereof.

In some embodiments, at least one A is —C═O. In some embodiments, each Ais —C═O.

In some embodiments, at least one D is O. In some embodiments, each D isO.

In some embodiments, each R¹ is, independently, hydrogen, methyl, ethyl,methoxy, ethoxy, halo, or haloC₁₋₃alkyl. In some embodiments, each R¹is, independently, hydrogen, methyl, methoxy, halo, or haloC₁₋₃alkyl. Insome embodiments, each R¹ is, independently, hydrogen, methyl, ormethoxy. In some embodiments, at least one R¹ is hydrogen. In someembodiments, each R¹ is hydrogen.

In some embodiments, each R² is, independently, hydrogen, methyl, ethyl,methoxy, ethoxy, halo, or haloC₁₋₃alkyl. In some embodiments, each R²is, independently, hydrogen, methyl, methoxy, or halo. In someembodiments, at least one R² is hydrogen. In some embodiments, each R²is hydrogen.

In some embodiments, each R³ is, independently, hydrogen, methyl, ethyl,methoxy, ethoxy, halo, or haloC₁₋₃alkyl. In some embodiments, each R³is, independently, methyl, methoxy, halo, or haloC₁₋₃alkyl. In someembodiments, each R³ is, independently, halo or haloC₁₋₃alkyl. In someembodiments, each R³ is, independently, haloC₁₋₃alkyl. In someembodiments, at least one R³ is trifluoromethyl. In some embodiments,each R³ is trifluoromethyl.

In some embodiments, each R⁴ is, independently, hydrogen, methyl, ethyl,methoxy, ethoxy, or haloC₁₋₃alkyl. In some embodiments, each R⁴ is,independently, hydrogen, methyl, methoxy, halo, or haloC₁₋₃alkyl. Insome embodiments, each R⁴ is, independently, hydrogen, methyl, methoxy,or halo. In some embodiments, at least one R⁴ is hydrogen. In someembodiments, each R⁴ is hydrogen.

In some embodiments, each A is, independently, —C═O or —C═S; each D is,independently, O or S; each R¹ is, independently, hydrogen, methyl,ethyl, methoxy, ethoxy, halo, halomethyl, or haloethyl; each R² is,independently, hydrogen, methyl, methoxy, halo, or halomethyl; each R³is, independently, C₁₋₃alkyl, C₁₋₃alkoxy, halo, or haloalkyl; and eachR⁴ is, independently, hydrogen, methyl, ethyl, methoxy, ethoxy, halo,halomethyl, or haloethyl.

In some embodiments, each A is, independently, —C═O or —C═S; each D is,independently, O or S; each R¹ is, independently, hydrogen, methyl,methoxy, halo, or halomethyl; each R² is, independently, hydrogen, halo,or halomethyl; each R³ is, independently, methyl, ethyl, methoxy,ethoxy, halo, halomethyl, or haloethyl; and each R⁴ is, independently,hydrogen, methyl, ethyl, methoxy, ethoxy, halo, halomethyl, orhaloethyl.

In some embodiments, each A is —C═O; each D is O; each R¹ is,independently, hydrogen, halo, or halomethyl; each R² is, independently,hydrogen or halo; each R³ is, independently, methyl, methoxy, halo, orhalomethyl; and each R⁴ is, independently, hydrogen, methyl, methoxy,halo, or halomethyl.

In some embodiments, each A is —C═O; each D is O; each R¹ is,independently, hydrogen or halo; each R² is, independently, hydrogen orhalo; each R³ is, independently, methyl, halo, or halomethyl; and eachR⁴ is, independently, hydrogen, methyl, halo, or halomethyl.

In some embodiments, each A is —C═O; each D is O; each R¹ is,independently, hydrogen or halo; each R² is, independently, hydrogen orhalo; each R³ is, independently, halo or halomethyl; and each R⁴ is,independently, hydrogen or halo.

In some embodiments, each A is —C═O; each D is O; each R¹ is,independently, hydrogen or halo; each R² is, independently, hydrogen orhalo; each R³ is, independently, methyl, halo, or halomethyl; and eachR⁴ is, independently, hydrogen, methyl, halo, or halomethyl.

In some embodiments, each A is —C═O; each D is O; each R¹ is,independently, hydrogen or halo; each R² is, independently, hydrogen orhalo; each R³ is, independently, halo or halomethyl; and each R⁴ is,independently, hydrogen, halo, or halomethyl.

In some embodiments, the compound is Compound A

or a pharmaceutically acceptable salt thereof.

Suitable examples of salts include, for example, hydrochloric acid andtrifluoroacetic acid.

The compounds of Formula I can contain one or more chiral centers and/ordouble bonds and, therefore, exist as stereoisomers, such as double-bondisomers (i.e., geometric isomers), enantiomers, or diastereomers.According to the invention, the chemical structures depicted herein, andtherefore the compounds of Formula I, encompass all of the correspondingcompound's enantiomers and stereoisomers, that is, both thestereomerically pure form (e.g., geometrically pure, enantiomericallypure, or diastereomerically pure) and enantiomeric and stereoisomericmixtures. Enantiomeric and stereoisomeric mixtures can be resolved intotheir component enantiomers or stereoisomers by well known methods, suchas chiral-phase gas chromatography, chiral-phase high performance liquidchromatography, crystallizing the compound as a chiral salt complex, orcrystallizing the compound in a chiral solvent. Enantiomers andstereoisomers can also be obtained from stereomerically- orenantiomerically-pure intermediates, reagents, and catalysts by wellknown asymmetric synthetic methods.

Compounds of Formula I further include hydrates and solvates.

Compounds containing an amine function can also form N-oxides. Areference herein to a compound that contains an amine function alsoincludes the N-oxide. Where a compound contains several amine functions,one or more than one nitrogen atom can be oxidized to form an N-oxide.Examples of N-oxides include N-oxides of a tertiary amine or a nitrogenatom of a nitrogen-containing heterocycle. N-Oxides can be formed bytreatment of the corresponding amine with an oxidizing agent such ashydrogen peroxide or a per-acid (e.g., a peroxycarboxylic acid) (see,Advanced Organic Chemistry, by Jerry March, 4th Edition, WileyInterscience).

In some embodiments, the compounds of Formula I are isolated and/orpurified.

The present invention also provides pharmaceutical compositionscomprising one or more of the compounds described above, or one or moresalts thereof, and a pharmaceutically acceptable carrier.

Suitable compositions include, but are not limited to, oral non-absorbedcompositions. Suitable compositions also include, but are not limited tosaline, water, cyclodextrin solutions, and buffered solutions of pH 3-9.

The compounds described herein, including Compound A, orpharmaceutically acceptable salts thereof, can be formulated withnumerous excipients including, but not limited to, purified water,propylene glycol, PEG 400, glycerin, DMA, ethanol, benzyl alcohol,citric acid/sodium citrate (pH3), citric acid/sodium citrate (pH5),tris(hydroxymethyl)amino methane HCl (pH7.0), 0.9% saline, and 1.2%saline, and any combination thereof. In some embodiments, excipient ischosen from propylene glycol, purified water, and glycerin.

In some embodiments, the excipient is a multi-component system chosenfrom 20% w/v propylene glycol in saline, 30% w/v propylene glycol insaline, 40% w/v propylene glycol in saline, 50% w/v propylene glycol insaline, 15% w/v propylene glycol in purified water, 30% w/v propyleneglycol in purified water, 50% w/v propylene glycol in purified water,30% w/v propylene glycol and 5 w/v ethanol in purified water, 15% w/vglycerin in purified water, 30% w/v glycerin in purified water, 50% w/vglycerin in purified water, 20% w/v Kleptose in purified water, 40% w/vKleptose in purified water, and 25% w/v Captisol in purified water. Insome embodiments, the excipient is chosen from 50% w/v propylene glycolin purified water, 15% w/v glycerin in purified water, 20% w/v Kleptosein purified water, 40% w/v Kleptose in purified water, and 25% w/vCaptisol in purified water. In some embodiments, the excipient is chosenfrom 20% w/v Kleptose in purified water, 20% w/v propylene glycol inpurified water, and 15% w/v glycerin in purified water.

In some embodiments, the formulation comprises 50 mg/mL Compound A in20% w/v Kleptose in purified water.

In some embodiments, the formulation can be lyophilized to a solid andreconstituted with, for example, water prior to use.

When administered to a mammal (e.g., to an animal for veterinary use orto a human for clinical use) the compounds of Formula I can beadministered in isolated form. Alternately, the compounds of Formula Ican be administered along with (i.e., as a combined formulation or asseparate formulations) with other antibiotics, such as, for example: 1)protein synthesis inhibitors including, but not limited to, amikacin,anisomycin, apramycin, azithromycin, blasticidine S, brefeldin A,butirosin, chloramphenicol, chlortetracycline, clindamycin,clotrimazole, cycloheximide, demeclocycline, dibekacin,dihydrostreptomycin, doxycycline, duramycin, emetine, erythromycin,fusidic acid, G 418, gentamicin, helvolic acid, hygromycin B, josamycin,kanamycin, kirromycin, lincomycin, meclocycline, mepartricin,midecamycin, minocycline, neomycin, netilmicin, nitrofurantoin,nourseothricin, oleandomycin, oxytetracycline, paromomycin, puromycin,rapamycin, ribostamycin, rifampicin, rifamycin, rosamicin, sisomicin,spectinomycin, spiramycin, streptomycin, tetracycline, thiamphenicol,thiostrepton, tobramycin, tunicamycin, tylosin, viomycin, andvirginiamycin; 2) DNA synthesis interfering agents including, but notlimited to, camptothecin, 10-deacetylbaccatin III, azacytidine,7-aminoactinomycin D, 8-quinolinol,

-   9-dihydro-13-acetylbaccatin III, aclarubicin, actinomycin D,    actinomycin I, actinomycin V, bafilomycin A1, bleomycin,    capreomycin, chromomycin, cinoxacin, ciprofloxacin,    cis-diammineplatinum(II) dichloride, coumermycin A1, L(+)-lactic    acid, cytochalasin B, cytochalasin D, dacarbazine, daunorubicin,    distamycin A, doxorubicin, echinomycin, enrofloxacin, etoposide,    flumequine, formycin, fumagillin, ganciclovir, gliotoxin,    lomefloxacin, metronidazole, mithramycin A, mitomycin C, nalidixic    acid, netropsin, nitrofurantoin, nogalamycin, nonactin, novobiocin,    ofloxacin, oxolinic acid, paclitaxel, phenazine, phleomycin,    pipemidic acid, rebeccamycin, sinefungin, streptonigrin,    streptozocin, succinylsulfathiazole, sulfadiazine, sulfadimethoxine,    sulfaguanidine purum, sulfamethazine, sulfamonomethoxine,    sulfanilamide, sulfaquinoxaline, sulfasalazine, sulfathiazole,    trimethoprim, tubercidin, 5-azacytidine, cordycepin, and formycin    A; 3) cell wall synthesis interfering agents including, but not    limited to, (+)-6-aminopenicillanic acid,    7-Aminodesacetoxycephalosporanic acid, amoxicillin, ampicillin,    azlocillin, bacitracin, carbenicillin, cefaclor, cefamandole,    cefazolin, cefmetazole, cefoperazone, cefotaxime, cefsulodin,    ceftriaxone, cephalexin, cephalosporin C, cephalothin, cephradine,    cloxacillin,-   D-cycloserine, dicloxacillin, D-penicillamine, econazole,    ethambutol, lysostaphin, moxalactam, nafcillin, nikkomycin Z,    nitrofurantoin, oxacillin, penicillic, penicillin G, phenethicillin,    phenoxymethylpenicillinic acid, phosphomycin, pipemidic acid,    piperacillin, ristomycin, and vancomycin; 4) cell membrane    permeability interfering agents (ionophores) including, but not    limited to, 2-mercaptopyridine, 4-bromocalcimycin A23187,    alamethicin, amphotericin B, calcimycin A23187, chlorhexidine,    clotrimazole, colistin, econazole, hydrocortisone, filipin,    gliotoxin, gramicidin A, gramicidin C, ionomycin, lasalocid A,    lonomycin A, monensin,    N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, narasin,    nigericin, nisin, nonactin, nystatin, phenazine, pimaricin,    polymyxin B, DL-penicillamine, polymyxin B, praziquantel,    salinomycin, surfactin, and valinomycin; 5) enzyme inhibitors    including, but not limited to, (+)-usnic acid, (±)-miconazole,    (S)-(+)-camptothecin,-   1-deoxymannojirimycin, 2-heptyl-4-hydroxyquinoline N-oxide,    cordycepin, 1,10-phenanthroline, 6-diazo-5-oxo-L-norleucine,    8-quinolinol, antimycin, antipain, ascomycin, azaserine,    bafilomycin, cerulenin, chloroquine, cinoxacin, ciprofloxacin,    mevastatin, concanamycin A, concanamycin C, coumermycin A1,    L(+)-lactic acid, cyclosporin A, econazole, enrofloxacin, etoposide,    flumequine, formycin A, furazolidone, fusaric acid, geldanamycin,    gliotoxin, gramicidin A, gramicidin C, herbimycin A, indomethacin,    irgasan, lomefloxacin, mycophenolic acid, myxothiazol,    N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, nalidixic acid,    netropsin, niclosamide, nikkomycin, N-methyl-1-deoxynojirimycin,    nogalamycin, nonactin, novobiocin, ofloxacin, oleandomycin,    oligomycin, oxolinic acid, piericidin A, pipemidic acid, radicicol,    rapamycin, rebeccamycin, sinefungin, staurosporine, stigmatellin,    succinylsulfathiazole, succinylsulfathiazole, sulfadiazine,    sulfadimethoxine, sulfaguanidine, sulfamethazine,    sulfamonomethoxine, sulfanilamide, sulfaquinoxaline, sulfasalazine,    sulfathiazole, triacsin C, trimethoprim, and vineomycin A1; and 6)    membrane modifiers including, but not limited to, paracelsin.

In some embodiments, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, or excipient with which acompound of Formula I is administered. Such pharmaceutical carriers canbe liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. The pharmaceutical carriers canalso be saline, gum acacia, gelatin, starch paste, talc, keratin,colloidal silica, urea, and the like. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents can be used.When administered to a human, the compounds of Formula I andpharmaceutically acceptable carriers can be sterile. Water is a suitablecarrier when the compound of Formula I is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical carriers also include excipients such as starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The present compositions, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents.

The compositions described herein can take the form of a solution,suspension, emulsion, tablet, pill, pellet, capsule, capsule containinga liquid, powder, sustained-release formulation, suppository, aerosol,spray, or any other form suitable for use. Examples of suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences, A. R. Gennaro (Editor) Mack Publishing Co.

In one embodiment, the compounds of Formula I are formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for administration to humans. Typically, compounds of Formula Iare solutions in sterile isotonic aqueous buffer. Where necessary, thecompositions can also include a solubilizing agent. Compositions forintravenous administration may optionally include a local anestheticsuch as lidocaine to ease pain at the site of the injection. Generally,the ingredients are supplied either separately or mixed together in unitdosage form, for example, as a dry lyophilized powder or water freeconcentrate in a hermetically sealed container such as an ampoule orsachette indicating the quantity of active agent. Where the compound ofthe invention is to be administered by infusion, it can be dispensed,for example, with an infusion bottle containing sterile pharmaceuticalgrade water or saline. Where the compound of Formula I is administeredby injection, an ampoule of sterile water for injection or saline can beprovided so that the ingredients may be mixed prior to administration.

The compounds of Formula I, and compositions comprising the same, can beadministered orally. Compounds and compositions for oral delivery can bein the form of, for example, tablets, lozenges, aqueous or oilysuspensions, granules, powders, emulsions, capsules, syrups, or elixirs.Orally administered compositions can contain one or more optionalagents, for example, sweetening agents such as fructose, aspartame orsaccharin; flavoring agents such as peppermint, oil of wintergreen, orcherry; coloring agents; and preserving agents, to provide apharmaceutically palatable preparation. Moreover, where in tablet orpill form, the compositions may be coated to delay disintegration andabsorption in the gastrointestinal tract thereby providing a sustainedaction over an extended period of time. Selectively permeable membranessurrounding an osmotically active driving compound are also suitable fororally administered compounds of Formula I. Oral compositions caninclude standard vehicles such as mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate, etc. Suchvehicles are suitably of pharmaceutical grade.

The pharmaceutical compositions can be in unit dosage form. In suchform, the composition can be divided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofthe preparations, for example, packeted tablets, capsules, and powdersin vials or ampules. The unit dosage form can also be a capsule, cachet,or tablet itself, or it can be the appropriate number of any of thesepackaged forms.

The present invention also provides methods of preparing Compound Acomprising:

1a) reacting (R)-(−)-N-Boc-3-pyrrolidinol with a strong base to form amixture; further reacting the mixture with2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene to form a compoundhaving Formula II

1b) reacting the compound of Formula II with an alcohol and a transitionmetal catalyst in the presence of hydrogen to form a compound of FormulaIII

1c) adding the compound of Formula III and pyrimidine-4,6-dicarboxylicacid to a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine andN-methylmorpholine to form a compound of Formula IV

1d) reacting the compound of Formula IV with N-Boc-guanidine butyricacid to form a compound of Formula V

1e) deprotecting the compound of Formula V to produce Compound A.

In some embodiments, in a) the strong base is NaH; and in b) thetransition metal catalyst is Pd/C and the alcohol is ethanol. Inparticular, this method is described below in more detail in Example 1.

The present invention also provides alternate methods of preparingCompound A comprising:

a) deprotonating (R)-3-Hydroxypyrrolidine-1-carboxylic acid tent-butylester, and reacting the resultant compound with2-chloro-1,3-dinitro-5-trifluoromethylbenzene to form(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester;

b) reducing(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester in the presence of an alcohol, a transition metalcatalyst, and hydrogen to form(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester;

c) coupling(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester with pyrimidine-4,6-dicarboxylic acid in thepresence of 1-[(3-(dimethylamino)-propyl)]-3-ethylcarbodiimidehydrochloride to form pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide};

d) reacting pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}with({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)pentanoicacid in the presence of phosphorous oxychloride to formpyrimidine-4,6-dicarboxylic acidbis-{[3-(5-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-pentanoylamino)-2-((R)-1-(tert-butoxycarbonyl)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide};

e) deprotecting pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-pentanoylamino)-2-((R)-1-(tert-butoxycarbonyl)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}to form crude pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-guanidino-pentanoylamino)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}tetrahydrochloride;and

f) purifying crude pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-guanidino-pentanoylamino)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}tetrahydrochlorideby reverse-phase chromatography.

In some embodiments, in b) the transition metal catalyst is Pd/C and thealcohol is ethanol. In particular, this method is described below inmore detail in Example 2.

The present invention also provides second alternate methods ofpreparing Compound A comprising:

a) deprotonating (R)-3-Hydroxypyrrolidine-1-carboxylic acid tent-butylester and further reacting the resultant compound with2-chloro-1,3-dinitro-5-trifluoromethylbenzene to form(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester;

b) reducing(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester in the presence of an alcohol, a transition metalcatalyst, and hydrogen, to form(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester;

c) coupling(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester with pyrimidine-4,6-dicarboxylic acid in thepresence of 1-[(3-(dimethylamino)-propyl)]-3-ethylcarbodiimidehydrochloride to form pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide};

d) reacting pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}with N-Cbz acid in the presence of thionyl chloride;

e) reducing the resultant compound of d) in the presence of an alcohol,a transition metal catalyst, and hydrogen;

f) reacting the resultant compound of e) with di-Boc pyrazole; and

g) deprotecting the resultant compound off) to produce Compound A.

In some embodiments, in b) and e) the transition metal catalyst is Pd/Cand the alcohol is ethanol. In particular, this method is describedbelow in more detail in Example 3.

One skilled in the art will be able to substitute suitable reagents forthe reagents recited in the methods described herein to produce CompoundA as well as additional compounds of Formula I.

The present invention also provides methods of preparing apharmaceutically acceptable salt of Compound A comprising:

a) reacting (R)-(−)-N-Boc-3-pyrrolidinol with a strong base to form amixture; further reacting the mixture with2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene to form a compoundhaving Formula II

b) reacting the compound of Formula II with an alcohol and a transitionmetal catalyst in the presence of hydrogen to form a compound of FormulaIII

c1) adding the compound of Formula III and pyrimidine-4,6-dicarboxylicacid to a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine andN-methylmorpholine to form a compound of Formula IV

c2) adding the compound of Formula III and pyrimidine-4,6-dicarboxylicacid to a mixture of 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimidehydrochloride (EDCl) and anhydrous pyridine to form a compound ofFormula IV

d) adding the compound of Formula IV with an N-Cbz acid to a solutioncomprising anhydrous pyridine, dimethylaminopropylamine, and any one ofthionyl chloride, POCl₃, (EtO)₂POCl, or oxalyl chloride to form acompound of Formula Va

e) hydrogenlysis of the Cbz group of the compound of Formula Va toproduce the compound of formula VI

f) protecting the compound of Formula VI to produce the compound offormula VII

g) deprotecting the compound of Formula VII to produce apharmaceutically acceptable salt of Compound A.

Preparation of Compounds of Formula I can Involve the Protection andDeprotection of various chemical groups. The need for protection anddeprotection, and the selection of appropriate protecting groups, can bereadily determined by one skilled in the art. The chemistry ofprotecting groups can be found, for example, in T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., Wiley &Sons, Inc., New York (1999), which is incorporated herein by referencein its entirety.

The present invention also provides methods of inhibiting the growth ofa microbe comprising contacting the microbe with one or more compoundsdescribed above, or a pharmaceutically acceptable salt thereof. In someembodiments, the compound of Formula I can act as an antiseptic agentfor cleansing surfaces, such as in, for example, kitchens and bathrooms.In these embodiments, the compound of Formula I can be formulated forsuch uses by procedures well known to the skilled artisan.

The present invention also provides methods of treating a mammal havinga microbial infection comprising administering to the mammal in needthereof an anti-microbial effective amount of one or more compoundsdescribed above, or a pharmaceutically acceptable salt thereof. In someembodiments, the mammal can be pre-diagnosed with a microbial infectionprior to treatment. In some embodiments, no formal diagnosis may havebeen made; in such embodiments, the mammal may be suspected of having amicrobial infection for which treatment is recognized as beingdesirable.

In one embodiment, “treatment” or “treating” refers to an ameliorationof a microbial infection, or at least one discernible symptom thereof;or to an amelioration of at least one measurable physical parameter, notnecessarily discernible by the patient; or to inhibiting the progressionof a microbial infection; or to delaying the onset of a microbialinfection.

In some embodiments, the microbe is, or the microbial infection is dueto, a gram-negative aerobe, a gram-positive aerobe, a gram-negativeanaerobe, a gram-positive anaerobe, or a yeast. In some embodiments, thegram-negative aerobe is selected from, but not limited to, Escherichiacoli, Citrobacter freundii, Citrobacter diverus, Citrobacter koseri,Enterobacter cloacae, Enterobacter faecalis, Klebsiella pneumonia,Klebsiella oxytoca, Morganella morganii, Providencia stuartii, Proteusvulgaris, Proteus mirabilis, Serratia marcescens, Acinetobacterhaemolyticus, Acinetobacter junii, Acinetobacter lwoffii, Haemophilusinfluenzae, Stenotrophomonas maltophilia, and Pseudomonas aeruginosa. Insome embodiments, the gram-positive aerobe is selected from, but notlimited to, Enterococcus faecalis, Enterococcus faecium, Mycobacteriumtuberculosis, Staphylococcus aureus, Staphylococcus pneumoniae,Staphylococcus epidermidis, Staphylococcus saprophyticus, Staphylococcuscolmii, Staphylococcus sciuri, Staphylococcus warneri, Streptococcusagalactiae, Streptococcus pyogenes, Streptococcus anginosus,Streptococcus mitis, and Streptococcus oxalis. In some embodiments, thegram-negative anaerobe is Bacteroides fragilis. In some embodiments, thegram-positive anaerobe is Clostridium difficile or Clostridiumperfringens. In some embodiments, the mycobacterium is Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium africanum,Mycobacterium canetti, or Mycobacterium microti. In some embodiments,the yeast is selected from, but not limited to, Candida albicans andCandida krusei.

In some embodiments, the microbe is an antibiotic-resistant strain ofbacteria, such as those recited in the Examples below.

The compounds of Formula I, or a pharmaceutically acceptable saltthereof, and compositions comprising the same, can be administered in avariety of routes, such as, for example, by infusion or bolus injection,and can be administered together with another biologically active agent,such as another antibiotic. Administration can be systemic or local.Various delivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc., and can be used toadminister a compound of Formula I. Routes of administration include,but are not limited to, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, epidural, oral, sublingual,intranasal, intracerebral, intravaginal, transdermal, rectally,pulmonary, by inhalation, or topically, particularly to the ears, nose,eyes, or skin. In some embodiments, suitable routes of administrationinclude intravenous, topical, and subcutaneous. The desired route ofadministration is left to the discretion of the practitioner, and willdepend, in part, upon the site of the microbial infection and medicalcondition of the mammal or human being treated. In most instances,administration can result in the release of the compounds of Formula Iinto the bloodstream.

In some embodiments, it may be desirable to administer one or morecompounds of Formula I, or a pharmaceutically acceptable salt thereof,locally to an area in need of treatment. This may be achieved, forexample, and not by way of limitation, by local infusion during surgery,topical application, e.g., in conjunction with a wound dressing aftersurgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, wherein the implant is of aporous, non-porous, or gelatinous material, including membranes, such assialastic membranes, or fibers.

The amount of a compound of Formula I, or a pharmaceutically acceptablesalt thereof, that will be effective in the treatment of a particularmicrobial infection will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques. Inaddition, in vitro or in vivo assays may optionally be employed to helpidentify optimal dosage ranges. The precise dose to be employed in thecompositions will also depend on the route of administration, and theseriousness of the infection, and should be decided according to thejudgment of the practitioner and each patient's circumstances. However,suitable dosage ranges for administration are generally about 0.001milligrams to about 200 milligrams per kilogram of body weight. In someembodiments, the dose is from about 0.01 milligrams to about 70milligrams per kilogram of body weight, or from about 0.1 milligrams toabout 50 milligrams per kilogram of body weight, or from about 0.5milligrams to about 20 milligrams per kilogram of body weight, or fromabout 1 milligram to about 10 milligrams per kilogram of body weight. Insome embodiments, the dose is about 5 milligrams per kilogram of bodyweight. The dosage amounts described herein refer to total amountsadministered; that is, if more than one compound of Formula I isadministered, the dosages correspond to the total amount of thecompounds of Formula I administered. Compositions can contain 10% to 95%active ingredient by weight. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.Such animal models and systems are well known in the art.

The present invention also provides one or more compounds describedabove, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition comprising one or more compounds describedabove, for treating a microbial infection.

The present invention also provides one or more compounds describedabove, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition comprising one or more compounds describedabove, for use in the manufacture of a medicament for the treatment of amicrobial infection.

The present invention also provides the use of one or more compoundsdescribed above, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition comprising one or more compounds describedabove, in the inhibition of growth of a microbe.

The present invention also provides the use of one or more compoundsdescribed above, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition comprising one or more compounds describedabove, in the treatment of a microbial infection in a mammal.

In order that the invention disclosed herein may be more efficientlyunderstood, examples are provided below. It should be understood thatthese examples are for illustrative purposes only and are not to beconstrued as limiting the invention in any manner. Throughout theseexamples, molecular cloning reactions, and other standard recombinantDNA techniques, were carried out according to methods described inManiatis et al., Molecular Cloning—A Laboratory Manual, 2nd ed., ColdSpring Harbor Press (1989), using commercially available reagents,except where otherwise noted.

EXAMPLES

Briefly, the results generated from the examples below indicate thatCompound A is active against Staphylococci spp. and other Gram-positiveand Gram-negative organisms. For example, susceptibility screens wereperformed against 150 isolates of S. aureus and coagulase-negativeStaphylococci with defined antibacterial susceptibilities to otherantimicrobials. Generally, MIC₉₀ values of 0.5 to 2.0 μg/ml have beenobtained in a screen of 150 Staphylococci organisms, and was notaffected by susceptibility phenotypes to other antibiotics. Serialpassage of methicillin-susceptible (MSSA ATCC 29213) and resistant (MRSAATCC 33591) strains of S. aureus at 0.5×MIC concentrations for 17passages did not result in any change in MIC values. Generally, CompoundA was bactericidal with time-kills ranging from 30 minutes to 6 hours.

Compound A was efficacious in vivo in a mouse thigh burden model againstMSSA 29213 and MRSA 33591 and in a mouse peritonitis/sepsis modelagainst MSSA 27660. In the mouse thigh burden model with MSSA 27660,Compound A achieved reductions 24 hours post-infection of up to 4¹⁰ incfu/thigh relative to untreated, infected control mice at dosages thatwere well-tolerated in repeat dose toxicity studies. Thus, robustefficacy against MSSA and MRSA was observed in a mouse thigh burdenmodel, and against MSSA in a rat thigh burden model and a mouseperitonitis model. Compound A was stable in the presence of plasma andisolated hepatocytes from multiple species.

Compound A was better tolerated in acute toxicity studies whenadministered by IV infusion. The MTD (IV bolus) for Compound A in themouse (30 mg/kg) is significantly higher than the static efficacy dosein the thigh burden model (2-4 mg/kg).

Compound A is currently in Phase 1 human clinical trials for developmentas an IV pan-Staphylococcal agent.

Example 1 Synthesis of Compound A

Step 1:

Sodium hydride (1.12 g, 60% in mineral oil, 28 mmol) was added inportion to anhydrous DMF (24 mL) solution of(R)-(−)-N-Boc-3-pyrrolidinol (5.0 g, 27.6 mmol) at room temperature. Theresulting mixture was stirred for an additional 15 minutes. This mixturewas then added dropwise to a DMF (20 mL) solution of2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene (7.45 g, 27.6 mmol) at0° C. The deep red solution was stirred at room temperature for 4 hours.The reaction was quenched by ice-water and extracted by ethyl acetate.The organic layer was washed by brine and water, and dried over Na₂SO₄.After removal of the solvent, the residue was purified by flash column(ethyl acetate/hexanes=¼, v/v). The yield was 54%.

Step 2:

(R)-tert-butyl3-(4-(trifluoromethyl)-2,6-dinitrophenoxy)pyrrolidine-1-carboxylate(4.84 g, 9.8 mmol) and Pd/C (0.78 g, 10% on carbon) and ethanol (140 mL)were placed in a Parr bottle. The mixture was flashed under hydrogenthree times and stirred under 40 psi hydrogen at room temperatureovernight. The mixture was filtrated through celite. The cake was washedtwice with ethanol (2×20 mL). The filtrate was evaporated under vacuum.An off-white solid was obtained and used as such for the subsequentreaction. The yield was 100%.

Step 3:

2-Chloro-4,6-dimethoxy-1,3,5-triazine (5.97 g, 34 mmol) was stirred inanhydrous THF (200 mL). N-Methylmorpholine (7.5 ml, 68 mmol) was added.The resulting mixture was stirred at room temperature for 30 minutes.Then, (R)-tert-butyl3-(2,6-diamino-4-(trifluoromethyl)phenoxy)pyrrolidine-1-carboxylate(10.84 g, 30 mmol) and pyrimidine-4,6-dicarboxylic acid (2.48 g, 14.8mmol) were added. The mixture was stirred at room temperature for 24hours. The solvent was evaporated completely in vacuum. Water (250 mL)was added and the mixture was stirred for 4 hours. After filtration, theyellow cake was washed with water (3×100 mL) and stirred in water (250mL) for 4 hours. The filtration and washing procedure was repeatedtwice. The solid was dried in the air and stirred in dichloromethane (20mL) for 30 minutes, followed by ultrasonic treatment for 1 hour. Afterfiltration, the yellow cake was quickly washed with cold dichloromethane(2×10 mL). The product (10.0 g, yield: 79.1%) was used as such forsubsequent reaction.

Step 4:

The starting material (6.5 g, 7.6 mmol), N-Boc guanidine butyric acid(10.9 g, 30.4 mmol) were stirred in anhydrous pyridine (40 mL) at 0° C.POCl₃ (2.78 mL, 30.4 mmol) in pyridine (4 mL) was added dropwise. Theresulting mixture was stirred at 0° C. for 1.5 hours. The reactionmixture was evaporated under vacuum. Water (140 mL) was added to theresidue. The mixture was extracted with ethyl acetate (260 mL). Theorganic layer was washed with brine (100 mL) and dried over Na₂SO₄.After evaporation, the residue was purified by column (Eluent: ethylacetate/hexanes/dichloromethane=1/1/1, v/v/v then 2%˜4% methanol indichloromethane). The yield was 29.1%. The R_(f) was same as thestandard sample which was characterized by NMR.

Step 5:

The starting material (3.4 g, 2.3 mmol) was stirred in 4N HCl in dioxane(34 mL) at room temperature overnight. The solvent was removed undervacuum. The residue was titrated in ether. The solid was filtered andpurified by C18 reverse-phase C18 column. A light yellow solid wasobtained as product with a purity of 98% (HPLC); LC-MS (M+1): 937.Yield: 51%.

Example 2 Synthesis of Compound A

Step 1: (R)-3-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester isde-protonated with potassium tert-butoxide (KOtBu) in tetrahydrofuran(THF). The resulting anion is reacted with2-chloro-1,3-dinitro-5-trifluoromethylbenzene in tert-butyl methyl ether(MTBE)/THF. When the reaction is complete, the reaction mixture isquenched with water and partitioned with more MTBE. The organic layer iswashed with brine and water and concentrated on a rotary evaporator. Thesolid concentrate is re-dissolved in methanol and re-precipitated withwater. The resulting precipitate is filtered and dried to afford(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester which can be used in the next step without furtherpurification.

Step 2: The product from Step 1 is dissolved in methanol andhydrogenated at 100-200 psi and 30-50° C. in the presence of 10% Pd/Cuntil the reduction is deemed complete by HPLC. The reaction mixture isfiltered through Celite. The filtrate is concentrated and dried toafford(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester which can be used in the next step without furtherpurification.

Step 3: The product from Step 2 is coupled withpyrimidine-4,6-dicarboxylic acid, in the approximate ratio of 2 moldiamine:1 mol diacid, in the presence of1-[(3-(dimethylamino)-propyl)]-3-ethylcarbodiimide hydrochloride (EDCI),in pyridine, under inert atmosphere, at ambient temperature. When thereaction is complete, the reaction mixture is diluted in water. Theresulting precipitate is separated and re-dissolved in MTBE. The MTBEsolution is washed with water, 0.2 N HCl, and brine, dried overanhydrous sodium sulfate, separated, and diluted in heptane. Theresulting precipitate is isolated by filtration and dried to affordpyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}which can be used in the next step without further purification.

Step 4: The product from Step 3 is reacted with 2.5-3 molar equivalentsof({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)pentanoicacid in pyridine, in the presence of phosphorous oxychloride, at atemperature around −5 to −10° C. The reaction is quenched with water attemperature of 15° C. The supernatant is separated from the amorphousprecipitate, which is re-dissolved in MTBE, washed with water and brine,dried over anhydrous sodium sulfate, separated, and diluted in heptane.The resulting precipitate is isolated by filtration and dried to affordpyrimidine-4,6-dicarboxylic acidbis-{[3-(5-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-pentanoylamino)-2-((R)-1-(tert-butoxycarbonyl)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}which is used in the next step without further purification.

Step 5: The product from Step 4 is de-protected (removal of sixtert-butoxycarbonyl groups) with 4M HCl/1,4-dioxane in formic acid atambient temperature. The reaction mixture is diluted with 1,4-dioxane.The resulting precipitate is filtered, washed with 1,4-dioxane and driedto afford crude pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-guanidino-pentanoylamino)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}tetrahydrochloride(crude Compound A). The crude product is further purified byre-precipitation from methanol solution with THF (50° C. to ambienttemperature) and/or re-precipitation from water/methanol solution withTHF at ambient temperature.

Step 6 (chromatographic purification): The final purification ofCompound A is achieved by reverse-phase chromatography (RP-HPLC) usingYMC ODS-AQ phase, 50 micron, 120 Angstrom, slurry packed into a ProChromdynamic axial compression column. The mobile phase is a gradient ofsolvent B in solvent A, where solvent A is water with 0.05%trifluoroacetic acid (TFA) and solvent B is acetonitrile with 0.05% TFA.Fractions containing purified product are concentrated by rotaryevaporation to afford Compound A as the trifluoroacetate salt. The finalhydrochloride salt form is re-generated by passing a water/methanolsolution of the trifluoroacetate salt through a Dowex 1×2-400 (Cl-form)ion-exchange column, collecting the API-containing eluate,concentrating, and drying.

Compound A bulk drug substance is stored at 2-8° C., protected fromlight and air, in amber HDPE containers or in double polyethylene bagsin a fiber drum.

Example 3 Synthesis of Compound A

Step 1: (R)-3-Hydroxypyrrolidine-1-carboxylic acid tert-butyl ester(compound 20) is de-protonated with potassium tert-butoxide (KOtBu) intetrahydrofuran (THF). The resulting anion is reacted2-chloro-1,3-dinitro-5-trifluoromethylbenzene (compound 2) in tert-butylmethyl ether (MTBE)/THF. When the reaction is complete, the reactionmixture is quenched with water and partitioned with more MTBE. Theorganic layer is washed with brine and water and concentrated on arotary evaporator. The solid concentrate is re-dissolved in methanol andre-precipitated with water. The resulting precipitate is filtered anddried to afford(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester which can be used in the next step without furtherpurification. This reaction has been carried out at a scale using 4.2 Kgof compound 2.

Step 2: Compound 21 is dissolved in methanol and hydrogenated at 100-200psi and 30-50° C. in the presence of 10% Pd/C until the reduction isdeemed complete by HPLC. The reaction mixture is filtered throughCelite. The filtrate is concentrated and dried to afford(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester (compound 22) with HPLC purity of 92.2%. Thereaction is done in four batches with a scale of 1.64 kg of compound 21for each batch.

Step 3: Compound 22 is coupled with pyrimidine-4,6-dicarboxylic acid(compound 8), in the approximate ratio of 2 mol diamine:1 mol diacid, inthe presence of 1-[(3-(dimethylamino)-propyl)]-3-ethylcarbodiimidehydrochloride (EDCI), in pyridine, under inert atmosphere, at ambienttemperature. When the reaction is complete, the reaction mixture isdiluted in water. The resulting precipitate is separated andre-dissolved in MTBE. The MTBE solution is washed with water, 0.2 N HCl,and brine, dried over anhydrous sodium sulfate, separated, and dilutedin heptane. The resulting precipitate is isolated by filtration anddried to afford pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}(compound 23) which can be used in the next step without furtherpurification. The reaction is carried out at a scale using 3.15 kg ofcompound 22.

Step 4: The solution of 3.66 g of DMAP in 60 ml anhydrous pyridine wascooled to 0° C. with ice bath. 3.60 g of thionyl chloride was addedslowly. The resulting solution was stirred for 10 minutes. The startingmaterial N-Cbz acid (7.53 g, 30 mmol), Cpd 23 (8.54 g, 10 mmol), wereadded to the solution respectively. The resulting mixture was stirred atRT for 4 hours. Water (500 mL) was added. After the mixture was stirredvigorously at room temperature for 2 hours, the solid was filtered andwashed with 250 mL of water. The solid was dissolved in ethyl acetate(300 mL). The organic layer was washed with 10% citric acid solution(100 mL) and brine (100 mL) and dried over Na₂SO₄. After evaporation,the residue was dissolved in 40 mL DCM, then 250 mL hexane was added.The precipitate was collected and dry under vacuum. 13.20 g of productwas obtained in 95% purity. Yield: 100%.

Step 5: Compound 26 (13.20 g) was dissolved in MeOH with 2 equiv. of 1 NHCl, and the catalyst Pd/C (10%) 1.0 g was added. The reaction mixturewas put on a Parr hydrogenator and shaken for 2 hours under 60 psi ofhydrogen. LCMASS showed no progress and another 1.0 g of catalyst wasadded. The reaction mixture was put on a Parr hydrogenator and shakenfor 3 hours under 60 psi of hydrogen. The mixture was filtered throughcelite to remove the catalyst. The filtrate was concentrated to drynesson a rotovap at 30° C. 11.50 g of product was obtained in 95% purity.Yield: 100%.

Step 6: Compound 27 (11.50 g, 10 mmol) was dissolved in 60 ml methanoland DCM (1:1). Then, 4.04 g triethylamine (40 mmol) was added. di-Bocpyrazole 9.3 gram (30 mmol) was added and the resulting mixture wasstirred at room temperature for 1 hour. After removing 95% of thesolvent, 300 mL water was added and the mixture was stirred vigorouslyfor 2 hours. The solid was filtered and washed with 300 mL water. Thesolid was dissolved in 300 mL ethyl acetate and dried over Na₂SO₄. Afterevaporating the solvent, the solid was dissolved in 40 mL DCM, then 500mL hexane was used to precipitate the product out. The solid wascollected and dried under vacuum. 13.0 gram of product was obtained in85% yield (90% purity).

Step 7: Compound 28 (1.5 g) was purified on 80 g silica gel column byusing a gradient of 10-88% EtOAc in DCM. Fractions with purity above 95%were collected, evaporated under vacuum, and dried. Recovery was 50-60%.Compound 28 with 95% purity (0.3 g) was dissolved and stirred in ethylacetate (3 mL) at room temperature (22° C.) under argon. HCl gas wasbubbling into the solution for 20 minutes. The color of the solution wasturning deep yellow as the bubbling was ongoing. The solid startedcrushing out in 15 minutes. The solution was stirred at room temperaturefor another 1 hour. Additional 4 mL of ethyl acetate was introduced intothe reaction mixture because of a loss of ethyl acetate. The mixture wasbubbling with HCl gas for 10 minutes. The mixture was stirred for 2.5hours. One third of the mixture was filtered and washed with ethylacetate. Two thirds of the mixture was stirred at room temperatureovernight. Then, 30 mL of ethyl acetate was added to the mixture. Afterfiltration, the cake was washed with ethyl acetate twice (2×140 mL) anddried. The solid was immersed in ethyl acetate (8 mL) and kept in afreezer. The reaction process was performed in 4 hours. Overnightstirring did not show much change. The purity of the final product was98% with one major impurity of 1.2%.

Example 4 Synthesis of Compound A

Step 1: To a nitrogen purged 4-necked 12 L RBF was added 305.32 g ofcompound 2, 700 mL of MTBE with stirring and the mixture was cooled inan ice/water bath. Compound 20 (212.43 g) was dissolved with thepotassium tert-butoxide (1.31 L of 1 M solution in THF) providing aslightly turbid mixture. This mixture was added to compound 21 solutionin the RBF over 86 minutes with stirring while maintaining <9.0° C.internal temperature. The reaction was removed from the cold bath 30minutes later and allowed to stir at ambient temperature for 15.5 hours.Upon stirring, several small ice chips were added with a temperaturedecrease from 21.4° C. to 18.4° C. Then water was added (1.5 L) and MTBE(1.5 L) and the mixture was stirred for 10 minutes. The mixture wasphase-split and separated in a 6 L reparatory funnel. The aqueous layerwas re-extracted with MTBE (500 mL). The organic layers were combinedand washed with 2:1 water/saturated brine (3×900 mL), and concentratedto a reddish/rust colored solid under reduced pressure. This solid wasdissolved in 2.45 L of MeOH and the solution was transferred to a 4 LErlenmeyer flask. Then water (1 L) was added with stirring, in portions,resulting in thick slurry. The mixture was covered and placed in arefrigerator (1-5° C.) for 16 hours. The solid was collected byfiltration and dried under vacuum. The dried product was a bright yellowpowder. Yield: 395.3 g, HPLC purity 94.0%.

The potassium tert-butoxide can be replaced with, for example, anyalkoxide, sodium hydride, potassium hydride, or any base that candeprotonate the hydroxyl of compound 20. Compound 2 can be substitutedat the 2 position by any halide.

Step 2: To a stainless steel 2 gal Parr stirrer unit was added thecatalyst Pd/C (10 wt %, 20 g), compound 21 from Step 1 (394.37 g) andthen 2 L of MeOH carefully with swirling. The vessel was charged withhydrogen and vented twice. The mixture was then stirred starting at 82psi hydrogen; the pressure dropped to 0 psi. The vessel was repeatedlyfilled to 62 psi, 28 psi and 36 psi, respectively, each time allowingthe pressure to go back to 0 psi (total uptake of 208 psi in 51minutes). The internal temperature started at 16° C. and the mixtureshowed a gradual but rapid exotherm to maximum of 38° C. The internaltemperature was maintained at 33-38° C. The vessel was pressurized to 49psi with uptake of 23 psi. The vessel was pressurized to 90 psi withuptake of 12 psi over 1 hour. The vessel was pressurized to 120 psi withuptake of 82 psi over 6 hours. The vessel was then re-pressurized to 51psi with uptake of 37 psi over 14.33 hours (the total uptake of hydrogenwas 362 psi). The reactor was dismantled and the mixture was filteredthrough a pad of celite 545 pre-moistened with MeOH (11.0 cm diameterBüchner funnel). The reactor and pad were rinsed with MeOH and the padwas suctioned until slow dripping and colorless filtrate (˜3.0 L totalvolume). The filtrate was further filtered through a fluted paper diskto remove some fine dark powders. The clear filtrate was transferred toa 5 L RBF and concentrated to orange/brown colored viscous oil which waschilled to 3-4° C. overnight, during which time the material partiallysolidified/crystallized. The material was warmed and further suctionedunder reduced pressure to form a solid rust/brown gummy/waxy hard solidchunk. To the RBF were added heptanes (2×700 mL) and MTBE (700 mL). Themixture was stirred with overhead mechanical stirring for 3.5 hours. Theliquid layer was decanted from the solid, the chunk was broken intosmaller pieces, the liquid was placed back into the RBF and thesuspension was stirred vigorously for 16.75 hours. Then the small piecesleft were further crushed using the end of a glass stir shaft and themixture was vigorously stirred for 70 minutes. The suspension wasfiltered through a tared fritted glass funnel using the filtrate tocomplete the transfer. The funnel was covered and vacuum dried with lowheat (41° C.) for 4 hours providing the product as a faint peachy/beigepowder. Yield 270.60 g, HPLC purity 98.5%.

The Pd/C catalyst can be replaced by, for example, any catalyst suitablefor the hydrogenation of a nitro group.

Step 3 (Option 1): 2-Chloro-4,6-dimethoxy-1,3,5-triazine (4.0 g) wasstirred in anhydrous THF (60 mL). N-Methylmorpholine (4.4 g) was added.The resulting mixture was stirred at room temperature for 30 minutes.Then compound 22 (7.2 g) and pyrimidine-4,6-dicarboxylic acid (1.68 g)were added. The mixture was stirred at room temperature for 24 hours.Then the solvent was evaporated completely in vacuum. Water (250 mL) wasadded and the mixture was stirred for 4 hours. After filtration, theyellow cake was washed with water (3×100 mL) and stirred in water (250mL) for 4 hours again. The filtration and washing procedure was repeatedtwice. Then the solid was dry in the air. The solid was dissolved in 15mL of DCM:Hexane:Acetone (5:5:1) solution. The mixture was kept at roomtemperature for 2 days. The solid was filtered and washed with 10 mL ofDCM:Hexane (1:1) solution twice. The recrystallization procedure wasrepeated one more time to give light yellow solid. Yield 70%, HPLCpurity 100%.

Step 3 (Option 2): EDCI (6.0 g) was stirred in anhydrous Pyridine (60mL). Then compound 22 (7.2 g) and compound 9 (0.56 g) were added. Themixture was stirred at room temperature for 2 hours. Then anotherportion pyrimidine-4,6-dicarboxylic acid (0.56 g) was added. After themixture was stirred at room temperature for another 2 hours, the thirdportion pyrimidine-4,6-dicarboxylic acid (0.56 g) was added. Theresulting mixture was stirred at room temperature for 24 hours. Then thesolvent was evaporated completely in vacuum. Water (250 mL) was addedand the mixture was stirred for 4 hours. After filtration, the yellowcake was washed with water (3×100 mL) and stirred in water (250 mL) for4 hours again. The filtration and washing procedure was repeated twice.Then the solid was dry in the air. The solid was dissolved in 15 mLDCM:Hexane:Acetone (5:5:1) solution. The mixture was kept at roomtemperature for 2 days. The solid was filtered and washed with 10 mLDCM:Hexane (1:1) solution twice. The recrystallization procedure wasrepeated one more time to give light yellow solid. Yield 70%, HPLCpurity 100%.

EDCI can be replaced with, for example, any amide coupling reagents thatgenerate acid anhydride or activated ester such as CDI, DCC, HOBt, HOAt,POCl₃.

Step 4: The solution of DMAP (3.66 g) in 60 mL anhydrous pyridine wascooled to 0° C. with ice bath. Thionyl chloride (3.60 g) was addedslowly. Then the resulting solution was stirred for 10 minutes. Thestarting material N-Cbz acid (7.53 g, 30 mmol), Cpd 5 (8.54 g, 10 mmol),were added to the solution respectively. The resulting mixture wasstirred at room temperature for 4 hours. Then water (500 mL) was added.After the mixture was stirred vigorously at room temperature for 2hours, the solid was filtered and washed with 250 mL water. The solidwas dissolved in ethyl acetate (300 mL). The organic layer was washedwith 10% citric acid solution (100 mL) and brine (100 mL) and dried overNa₂SO₄. After evaporation, the residue was dissolved in 40 mL DCM, then250 mL hexane was added. The precipitate was collected and dry undervacuum. 13.20 g product was obtained in 95% purity. Yield: 100%.

The thionyl chloride can be replaced with, for example, POCl₃,(EtO)₂POCl, or oxalyl chloride.

Step 5: Compound 26 (13.20 g) was dissolved in MeOH with 2 equiv. of 1 NHCl, and the catalyst Pd/C (10%) 1.0 gram was added. The reactionmixture was put on a Parr hydrogenator and shaking for 2 hours under 60psi of hydrogen. LCMASS showed no progress and another 1.0 gram catalystwas added. The reaction mixture was put on a Parr hydrogenator andshaking for 3 hours under 60 psi of hydrogen. The mixture was filteredthrough celite to remove the catalyst. The filtrate was concentrated todryness on a rotovap at 30° C. 11.50 g product was obtained in 95%purity. Yield: 100%.

The Pd/C catalyst can be replaced with, for example, any catalystsuitable for the hydrogenlysis of CBZ group.

Step 6: Compound 27 (11.50 g, 10 mmol) was dissolved in 60 mL methanoland DCM (1:1). Then 4.04 g triethylamine (40 mmol) was added. di-Bocpyrazole 9.3 gram (30 mmol) was added and the resulting mixture wasstirred at room temperature for 1 hour. After removing 95% of thesolvent, 300 mL water was added and the mixture was stirred vigorouslyfor 2 hours. The solid was filtered and washed with 300 mL water. Thesolid was dissolved in 300 mL ethyl acetate and dried over Na₂SO₄. Afterevaporating the solvent, the solid was dissolved in 40 mL DCM, then 500mL hexane was used to precipitate the product out. The solid wascollected and dried under vacuum. 13.0 g product was obtained in 85%yield (95% purity).

The di-Boc pyrazole can be replaced with, for example, isourea or di-Bocisourea.

Step 7: Compound 28 (1.17 kg, 0.76 mol) was dissolved in 24 L of EtOAc,followed by addition of 281 mL of water. HCl gas was added to thesolution while the temperature of reaction was controlled below 45° C.by adjust addition speed. The total reaction time is 5 hours among which1.5 hour is the time of HCl addition. HPLC indicated the startingmaterial is less than 1% and the precipitated product was collected byfiltration under nitrogen. The solid was rinsed with EtOAc, trituratedwith MeOH/THF (1:1) and dried under vacuum. Yield 84%.

The HCl/EtoAC can be replaced with, for example, HCl/dioxane.

Example 5 Antimicrobial Activity Vs. Gram-Positive Clinical Isolates(Table 1A) and Gram-Negative Clinical Isolates (Table 1B)

Compound A was evaluated in vitro in accordance with defined CLSIdocuments specific to the organisms (aerobic, anaerobic or yeast) testedin this study. Ampicillin, ceftazidime, cefuroxime, ciprofloxacin,linezolid, and vancomycin were tested alongside as comparator agents foraerobic bacteria; clindamycin and metronidazole were tested ascomparators for anaerobes; fluconazole was tested as a comparator foryeast isolates. Stock solutions of Compound A were prepared in dimethylsulfoxide (DMSO). Ampicillin, ceftazidime, cefuroxime, ciprofloxacin,linezolid, vancomycin, metronidazole, clindamycin, and fluconazole wereprepared each according to its manufacturer's guideline.

Aerobes (M7-A7)1

Minimum inhibitory concentrations (MICs) in μg/ml were determinedaccording to CLSI guideline M7-A7 by broth microdilution. All aerobeswere tested using Mueller-Hinton broth medium with the exception ofStreptococcus spp., which were tested using cation-adjustedMueller-Hinton broth supplemented with 2-5% lysed horse blood. Resultsare shown in Table 1A and Table 1B.

TABLE 1A Organism MIC (μg/mL)* 2-3 isolates/organism Gram-PositiveCompound A Linezolid Vancomycin Ceftazidime Entero. faecalis 1 1-2 1 >64Entero. faecium (VRE) 1 1-2 >128    >64 Staph. aureus (MRSA) 0.5-1   1-20.5-1   32 Staph. spidermidis 0.25-0.5  0.5-1   2 16-32 Staph.saprophyticus 0.25-0.5  1-2 1-2   32->64 Staph. spp. (coagulase−)0.25-0.5  1 1-2 16-32 Strept. agalactiae 2 1   0.5 0.5 Strept.pneumoniae 4-8 1   0.5 0.25 Strept. pyogenes 1-4 1   0.5 0.12 Strept.viridians 2-8 1 0.5-1   0.5-4   *Broth microdilution assays performedaccording to standard CLSI guidelines.

TABLE 1B Organism MIC (μg/mL)* 2-3 isolates/organism Gram-NegativeCompound A Ceftazidime Linezolid Vancomycin Citrobacter freundi 2-40.25-2   >16 >128 Citrobacter koseri 1-2 0.12-0.25 >16 >128 Enterobactercloacae 0.5-4   0.25 >16 >128 Escherichia coli 1-2 0.06 >16 >128Klebsiella oxytoca 2-8 0.06-0.12 >16 >128 Klebsiella pneumoniae 1-20.06-0.12 >16 >128 Morganella morganii    2->64  2-16 >16 >128 Proteusmirabilis   64->64 0.03-0.06 >16 >128 Proteus vulgarisi   64->640.12 >16 >128 Providencia stuartii 16-64 0.12-0.64 >16 >128Acinetobacter spp.  4  2-64 >16 128->128 Pseudomonas aeruginosa 321-8 >16 >128 Serratia marcescens 32 0.12-0.25 >16 >128 Stenotrophomonas   8->64 4-8 >16 32-128 maltophilia Haemophilus influenzae 4-8 0.06-0.1216->16   128 *Broth microdilution assays performed according to standardCLSI guidelines.

Compound A exhibited broad coverage against the Gram-positive pathogenswith S. aureus and coagulase-negative Staphyloccal species showing thelowest MICs. Compound A was active against the Gram-negative pathogensbut overall coverage was less than for the Gram-positive organisms.

Example 6 MICs with Staphylococcus Species with Defined ResistancePhenotypes

Evaluation of the susceptibility profiles of Compound A against selectedisolates was carried out in vitro by broth microdilution methodologyusing Mueller-Hinton broth medium according to CLSI document M7-A7. CLSIinterpretive breakpoints were applied where applicable as directed byCLSI document M100-S17. Results are shown in Table 2.

TABLE 2 VRSA/VISA Drug- VRSA/VISA LZD-NS DAP-NS DAP-NS suscept. OXA-ROXA-R OXA-R OXA-R OXA-R isolates 59 69 7 5 5 3 Compound A 0.25-1 0.25-20.5-1 0.5-1 0.5-2 0.5-1 MIC range *Broth microdilution assays performedaccording to standard CLSI guidelines. OXA-R: oxacillin-resistant; VRSA:vancomycin-resistant S. aureus; VISA: vancomycin intermediate S. aureus;LZD-NS: linezolid non-suscepetible; DAP-NS: daptomycin non-susceptible.

Compound A was active in vitro against all isolates of S. aureus andcoagulase-negative staphylococci, including isolates of S. aureus withcharacterized resistance to daptomycin, linezolid, and vancomycin(last-line therapeutics for the treatment of resistant S. aureus such asMRSA). Against S. aureus isolates, there was no alteration in activityagainst resistant isolates relative to susceptible isolates. Againstcoagulase-negative staphylococci, activity was not affected byresistance to methicillin.

Example 7 Cytotoxicity and Selectivity

Cytotoxicity of Compound A was evaluated in a colorimetric assay using atransformed human liver cell line (HepG2, HB-8065) and an embryonicmouse cell line (NIH/3T3 cells, CRL-1658). This assay measures thebioreduction of a novel tetrazolium compound to a soluble formazanproduct by viable cells. HepG2 cells were seeded in 96 well plates at2×10⁴ cells/well in MEM medium with 10% fetal bovine serum (FBS) 24hours prior to use. NIH/3T3 cells were seeded in 96 well plates at 2×10⁴cells/well in DMEM medium with 10% bovine calf serum (BCS) 24 hoursprior to use. Cell monolayers were rinsed in serum-free media andincubated for one hour with Compound A in serum-free media. Afterincubation, the media was replaced with serum supplemented media andlive cells were measured using the Cell Titer 96 AqueousNon-Proliferation Assay kit (Promega, Madison, Wis.). EC₅₀ values weredetermined using a four parameter logistic equation:Y=Bottom+(Top-Bottom)/(1+10^((LogEC₅₀−X)*HillSlope)).

Cytotoxicity of Compound A was also evaluated in a hemolysis assay usinghuman erythrocytes. Pooled whole human blood was centrifuged to separatethe red blood cells (RBC). The isolated RBCs were rinsed and diluted inTris-buffered saline (TBS buffer, pH 7.4) to obtain a 0.22% RBC stocksuspension. 5 μL of Compound A stock solution was added to 45 μL of RBCsuspension and incubated with shaking for 1 hour at 37° C. At theconclusion of the incubation time, samples were centrifuged and 30 μL ofthe supernatant was added to 100 μL of water. OD₄₁₄ measurements wereread for hemoglobin concentration. The bee venom peptide melittin wasused as a positive control. EC50 values were determined as describedabove. Results are shown in Table 3.

TABLE 3 MIC or MIC₉₀* Cytotoxicity Selectivity (μg/mL) (EC₅₀ μg/mL)(EC₅₀/MIC) Compound S. aureus RBCs 3T3 HepG2 RBCs 3T3 HepG2 Compound1.0* >500 430 1,031 >500 430 1,031 A melittin 2 2 4 1 1 2 0.5 Compound Ademonstrates great overall selectivity.

Example 8 Time-Kill Vs S. Aureus (ATCC 27660)

Time-kill studies of Compound A versus E. coli ATCC25922, E. coli (labstrain) D31, and S. aureus ATCC27660 were determined in a standardprotocol by measuring the time it takes to reduce the initial inoculums3 log units. Three ml of cation-adjusted Mueller-Hinton medium wasinoculated with 20 μL of frozen bacterial stock and incubated at 37° C.on a shaker platform (250 rpm) overnight. The suspension was diluted toapproximately 5×10⁵ cfu/mL and treated with 2×, 5×, 10×, and 20×MIC(MIC=1 μg/mL). Compound A stock solution was prepared at 10 mg/mL inDMSO. Time points were collected and viable bacteria were counted on MHAgar plates after an 18 hour incubation. Studies examining time-killkinetics of Compound A against S. aureus ATCC 27660 at 2×MICconcentrations show that reductions of 3 log₁₀ units in the initialinoculum occur in 5 hours. No re-growth is observed in the cultures over72 hours at 1×MIC concentrations. See, FIG. 1A and FIG. 1B.

Example 9 Serial Passage Resistance in MSSA (ATCC 29213) and MRSA (ATCC33591)

Frozen bacterial stocks (20 μL) of S. aureus ATCC29213 ormethicillin-resistant S. aureus (MRSA ATCC 33591) were inoculated into 3mL cation-adjusted Mueller-Hinton medium and incubated at 37° C. on ashaker platform (250 rpm) overnight. The suspension was diluted toapproximately 5×10⁵ cfu/mL and inoculated into a polypropylene (Costar)96-well round bottom plate (90 μL volumes). Compound stock solutions ofCompound A and norfloxacin (Sigma Aldrich, St. Louis, Mo.; Catalogue#N9890) were prepared in DMSO and serial two-fold dilutions of compoundwere made in 0.01% acetic acid, 0.2% bovine serum albumin directly inthe wells of the polypropylene plate at 10 μL/well. Final concentrationsof Compound A were 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.19,0.098, 0.049, and 0.024 μg/mL. Final concentration ranges of norfloxacinwere 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.19, 0.098, and0.049 μg/mL. DMSO concentrations did not exceed 1% in the assay. Allsamples were performed in triplicate. Following a 24 hour incubation at37° C., cell growth was assessed by observing the presence of“acceptable growth”, defined by CLSI as a ≧2 mm button or definiteturbidity. The MIC wells were defined as the lowest concentration whereacceptable growth was not observed. For serial passage, 50 μL aliquotswere taken from 2 of 3 replicate wells at 0.5×MIC and combined into 900μL of fresh cation-adjusted Mueller-Hinton medium. The OD₆₀₀ wasmeasured and the cell suspensions were inoculated into polypropylene96-well round bottom plates (90 μL volumes) at approximately 5×10⁵cfu/mL. Ten μL of compound stock solutions were added previously to thewells to achieve the concentration ranges for each compound describedabove. All samples were performed in triplicate. The plates wereincubated for 24 hours at 37° C. This process was repeated for a totalof 17 passages and MIC values were recorded at each passage.

Passage of S. aureus with norfloxacin was associated with a significantrise in MIC values by passage 3 (4 doubling dilutions) for both MSSA andMRSA that reached 128-fold and 64-fold increases, respectively, bypassage 15. Conversely, there was no change in the MICs for Compound Aagainst MSSA ATCC 29213 or MRSA ATCC 33591 over the entire 17 passagetime course. See, FIG. 2.

Example 10 In Vitro Metabolic Stability of Compound A—Blood Plasma

Pooled plasma samples from human (mixed gender), rat (mixed breed andgender) and dog (mixed breed and gender) were incubated with Compound A(5 μM) at 37° C. for 0 and 60 minutes (duplicate samples). Incubationswere terminated by addition of ice-cold precipitation solvent(acetonitrile:glacial acetic acid, 9:1 v/v). Supernatants were dilutedwith equal volume of 0.1% formic acid and analyzed by HPLC-MS/MS. Plasmastability is reported as % parent compound at 60 minutes relative toamount of parent at 0 minutes. Results are shown in Table 4.

TABLE 4 Species Plasma Stability (%) Human 96 Rat (mixed breeds) 102 Dog(mixed breeds) 100

There is little to no loss of Compound A in human, rat and dog plasmafollowing 1 hour incubation at 37° C., indicating high plasma stability.There was also little to no loss of Compound A in humans, cynomolgusmonkeys, and rabbits, (data not shown).

Example 11 Efficacy of Compound A in the Mouse Thigh Burden Model

Female 6-7-week old CD-1 mice were made neutropenic withcyclophosphamide (150 mg/kg, i.p.) on days 4 and 1 before i.m.inoculation with S. aureus (ATCC 13709). S. aureus inoculum was preparedby transferring colonies from 18-20-hour tryptic soy agar (TSA) culturesto sterile PBS. The density was adjusted to approximately 10⁶ cfu/mLwith the aid of a spectrophotometer, and the inoculum concentration wasdetermined by the dilution plate count method. Mice were inoculated byinjecting each posterior thigh with 0.1 mL of inoculum. Compound A wasgiven to separate groups of mice (4 females/group) by i.v. bolus dosesof 1 or 2 mg/kg/dose at 1 and 5, 1 and 9, or 1 and 13 hours postinoculation as shown in Table 5. A separate control group of 4 micereceived the inoculum without antibiotic treatment. Compound A wasdissolved 50%/50% v/v sterile USP purified water/PBS. Thighs wereharvested at 25 hours after inoculation. Thigh muscle and bone tissuewere homogenized, aliquots of serial dilutions were plated on TSA andincubated at 37° C. for 20 hours, and colony counts were obtained tocalculate cfu/thigh. The parameters are shown in Table 5.

TABLE 5 Thigh Dose Total Treatment Harvest Group (mg/kg/ Dose Volume (hrafter (hr after No. No. Treatment dose) (mg/kg) (ml/kg) inoculation)inoculation) Mice 1 Inoc. control NA NA NA NA 25 4 2 Compound A 1 2 4 1and 5 25 4 3 Compound A 2 4 4 1 and 5 25 4 4 Compound A 1 2 4 1 and 9 254 5 Compound A 2 4 4 1 and 9 25 4 6 Compound A 1 2 4  1 and 13 25 4 7Compound A 2 4 4  1 and 13 25 4

Compound A was most effective in reducing the bacterial population ininoculated thighs when administered at 2 mg/kg/dose at either 1 and 5 or1 and 9 hours after inoculation. Bacterial reductions in these 2 groupswere 3.96 and 3.93 logs lower, respectively, than those of theinoculated control group. See, FIG. 3.

Example 12 Efficacy Vs. Vancomycin in the Rat Thigh Burden Model

For each experiment, female 8-9-week old femoral vein cannulatedCrl:CD(SD) rats were made neutropenic with cyclophosphamide (150 mg/kg,i.p.) on days 4 and 1 before i.m. inoculation with S. aureus (ATCC13507). A suspension of S. aureus was prepared from colonies obtainedfrom an overnight culture, placed in PBS, and adjusted to approximately10⁷ cfu/mL with the aid of a spectrophotometer. Each rat was injected0.2 mL of inoculum into the thigh muscle of the right hind leg. Thighswere harvested at 25 hours after inoculation and processed to determinecfu/thigh. Compound A was given by i.v. bolus injection into a tail veinor 1-hour i.v. infusion, or 4-hour i.v. infusion via the femoral veincannulae at different time intervals following inoculation. Separateinoculation control groups were included in each experiment, andvancomycin groups were included as comparative agents in the first andsecond experiments. Each group, including the controls and comparativeagent, consisted of 4 rats.

For Compound A, i.v. bolus (10 mg/kg/dose, 20 mg/kg total dose) and 1hour i.v. infusions (10 mg/kg/dose, 20 mg/kg total dose) reduced thebacterial load by 3.2 and 3.0 logs, respectively, in comparison toinoculated controls. Reductions relative to inoculum levels at 1 hourpost-infection were approximately 2.2 to 2.0 logs, respectively.Efficacy was comparable to vancomycin. See, FIG. 4.

Example 13 Efficacy Compound A in Mouse Sepsis Model: S. aureusInfection

Sterile saline, vancomycin, or Compound A were administered to separategroups of 8-week old female CD-1 mice (8 mice/group) 1 and 7 hours afteri.p. injections of S. aureus (ATCC 13709, 5×10⁷ cfu/mL in 5% mucin, 0.5mL/mouse). Compound A was dissolved in 50%/50% v/v sterile USP purifiedwater/TBS. A suspension of S. aureus was prepared from coloniestransferred from the TSA plate to sterile PBS. An aliquot of the stocksuspension was added to 5% mucin for a final concentration of about5×10⁷ cfu/mL. Study design and doses are shown in Table 6. The mice wereobserved for 6 days following inoculation for mortality.

TABLE 6 Test Compound Dose Route Injection (mg/ Total of Test Schedulekg/ Dose Com- Volume (hr after No. Treatment dose) (mg/kg) pound (ml/kg)inolulation) Mice Inoc. control NA NA NA NA NA 8 Vancomycin 10 10 s.c.10  1 8 Compound A 3 6 i.v. 4 1 & 7 8 Compound A 5 10 i.v. 4 1 & 7 8Compound A 10 20 i.v. 4 1 & 7 8

Dose-dependent efficacy was observed with Compound A that was comparableto the vancomycin treatment group. All untreated mice died within thefirst day of treatment. At the 2×5 and 2×10 mg/kg doses of Compound A,full protection was achieved with Compound A. See, FIG. 5.

Example 14 Acute Toxicity Studies—Maximum Tolerated Doses

Maximum tolerated dose (MTD) determinations were made inascending/descending dose studies in mice and rats. Compound A wasadministered by either i.v. bolus injection in the tail vein of mice andrats or by i.v. infusion via catheter in the femoral vein of rats. Ateach dose, two to three animals were administered compound and clinicalsigns were recorded over a 4 to 7 day period. Gross necropsy wasperformed at the conclusion of the study. Results are shown in Table 7.

TABLE 7 MTD (mg/kg) Dosage Compound A mouse rat i.v. bolus 30 N.D. i.v.infusion - 1 hour N.D. >24

The MTD for Compound A in the rat was >24 mg/kg when administered byi.v. infusion for 1 hour.

Example 15 Pharmacokinetics of Compound A in Rats

Crl:CD (SD) rats were administered Compound A by i.v. bolus injection atthe indicated dosages. Plasma was prepared from blood samples taken at 9time points (n=3) over 28 hours. Compound levels were determine byHPLC-MS/MS. All animals were fitted with two jugular vein cannula (JVC),one each for dose administration and blood collection. Each route ofadministration was dosed as N=3. Animals were supplied with a commercialrodent diet and water ad libitum. Each rat received a bolus dosed viathe appropriate route of administration at time zero on the day ofdosing. Blood sampling times are shown in Table 8.

Each blood sample was collected from the rats via a JVC and placed intochilled polypropylene tubes containing sodium EDTA as an anticoagulant.Samples were centrifuged at a temperature of 4° C. and at a speed of13,000 rpm for 5 minutes. Samples were maintained chilled throughoutprocessing. Each plasma sample was then transferred into labeledpolypropylene tubes, placed on dry ice, and stored in a freezer set tomaintain −60° C. to −80° C.

Plasma study samples were extracted and analyzed using a previouslydeveloped method. A single standard curve and six replicates of qualitycontrol samples at three concentrations were extracted using DMSOcontaining 0.1% formic acid. Plasma samples (50 μL) were added to 150 μLsolvent and centrifuged. Supernatants were analyzed by LC/MSMS using aPerkin Elmer series 200 micropump and PE Sciex API4000 Electrospray massspectrometer. Standard curves were prepared at concentrations of 10000,5000, 1000, 500, 250, 100, 50 and 25 ng/mL. Quality control samples wereprepared at concentrations of 5000, 500, and 50 ng/mL. The standardcurve and quality control samples were prepared from independentlyprepared stock solutions. At least ⅝ of standards must have accuracywithin ±15%, except at the LLOQ where ±20% is acceptable. Two thirds ofthe batch QCs must have accuracy within ±15% of nominal, and at leastone QC must pass at each level in order for the run to be accepted.

Individual plasma concentration versus time data for Compound A wassubjected to non-compartmental analysis using the pharmacokineticprogram WinNonlin v4.1. Plasma concentrations below the limit ofquantitation (25 ng/ml) were assigned a value of zero forpharmacokinetic analysis. Nominal dosing concentrations were used in allcalculations.

TABLE 8 Dose Dosing (mg/kg, Solution Dosing Sampling Treatment TestAnimals free Conc. Volume Time Group Compound (N) base) (mg/mL) (mL/kg)Vehicle Points 1 Compound A 3 5 1.25 4 Tris- Predose, Buffered 2, 5, 15,Saline, 30 pH 7.4 minutes, 1, 2, 4 and 8 hours postdoseResults are shown below in Table 9.

TABLE 9 PK parameter Compound A (5 mg/kg, i.v. bolus) C_(max) (μg/mL)89.2 T_(1/2) (hours) 3 V_(D) (mL/kg) 110 C_(L) (mL/hr/kg) 28

The plasma half-life for Compound A in rat plasma was significantly longand clearance values are low.

Example 16 Formulations

The saturation solubility of Compound A in various excipients wasinvestigated at 25° C. and the results are reported in Table 10(saturation solubility of Compound A as the free base).

TABLE 10 Compound A (free base) saturation Functional solubility at 25°C. Excipient Category (mg/mL) Purified Water Control 65 Propylene GlycolCosolvents 90 PEG 400 18.5 Glycerin 53 DMA 0.60 Ethanol 1.13 Benzylalcohol 1.83 Citric Acid/Sodium Citrate Buffers 65.5 (pH 3) CitricAcid/Sodium Citrate 11.2 (pH 5) Tris(hydroxymethyl)amino 61.4 methaneHCl (pH 7.0) 0.9% Saline Diluent N/A 1.2% Saline N/A N/A: Not availableas the formulation formed a viscous yellow gel

Preliminary investigations indicated that benzyl alcohol, ethanol andDMA were poor vehicles for Compound A with a saturation solubility valueof 1.83, 1.13 and 0.60 mg/mL respectively. On the other hand, goodsaturation solubility of 90 mg/mL, 65 mg/mL and 53 mg/mL were achievedin propylene glycol, purified water and glycerin and were consequentlyinvestigated further. Good solubility values were achieved at pH 3 and7.4 with values of 65.5 and 61.4 mg/ml respectively. The solubilityhowever, appeared to drop at pH5 with a value of 12.1 mg/mL. Thesaturation solubility of Compound A in 0.9% and 1.2% sodium chloridesolution could not be detected as the formulation formed a viscousyellow gel.

The saturation solubility of Compound A in various multi-componentsystems was investigated at 25° C. and the results are reported in Table11 (saturation solubility of Compound A as the free base).

TABLE 11 Compound A (free base) saturation solubility at 25° C.Excipient Diluent (mg/mL) 20% w/v propylene glycol saline N/A 30% w/vpropylene glycol saline N/A 40% w/v propylene glycol saline N/A 50% w/vpropylene glycol saline N/A 15% w/v propylene glycol purified water 64.930% w/v propylene glycol purified water 59.1* 50% w/v propylene glycolpurified water 74.7 30% w/v propylene glycol purified water 43.9 and 5w/v ethanol 15% w/v glycerin purified water 63.5 30% w/v glycerinpurified water 63.1 50% w/v glycerin purified water 56.8 20% w/vKleptose purified water 79.7 40% w/v Kleptose purified water 102.0 25%w/v Captisol purified water 64.3 N/A: Not available as the formulationformed a viscous yellow gel *The formulation gelled duringcentrifugation but liquefied on standing.

The formulations which exhibited suitable results included 50% w/vpropylene glycol and 15% w/v glycerin in purified water with asaturation solubility value at 25° C. of 74.7 mg/mL and 63.5 mg/mLrespectively. Good saturation solubility was also achieved with variouscomplexing agents with values of 79.7, 102.0 and 64.3 mg/mL for 20% w/vKleptose, 40% w/v Kleptose and 25% w/v Captisol respectively. Resultsalso indicated that addition of Compound A to 20-50% w/v propyleneglycol in saline resulted in the formation of a viscous yellow gel andconsequently could not be analyzed by UV. However, the gellingphenomenon was concentration dependent and was observed in formulationsas the Compound A concentration in the formulation approached thesaturation solubility value of the drug. Additionally, the gellingprocess could be easily reversed by adding a small volume of theexcipient formulation or a few drops of ethanol. Addition of 5% w/vethanol into the formulation did not inhibit the gelling phenomenon butit was still easily reversible and able to be analyzed by UVspectrophotometry.

Following an evaluation of preliminary excipient screening data, threesuitable formulations were chosen for further formulation development.These formulations are 20% w/v Kleptose solution, 20% w/v propyleneglycol in purified water, and 15% w/v glycerin in purified water.

The formulation of 50 mg/mL Compound A in 20% w/v Kleptose was selectedin phase I clinical trial. In addition, solutions of Compound A inwater, Kleptose, or Mannitol can be aliquoted out and lyophilized to asolid. The solid can be reconstituted with water before use.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

1. A compound of Formula I

wherein: each A is, independently, —C═O, or —C═S; each D is,independently, O or S; each R¹ is, independently, hydrogen, methyl,ethyl, methoxy, ethoxy, halo, halomethyl, or haloethyl; each R² is,independently, hydrogen, methyl, methoxy, halo, or halomethyl; each R³is, independently, C₁₋₃alkyl, C₁₋₃alkoxy, halo, or haloalkyl; and eachR⁴ is, independently, hydrogen, methyl, ethyl, methoxy, ethoxy, halo,halomethyl, or haloethyl; or a pharmaceutically acceptable salt thereof.2. The compound or salt of claim 1 wherein each A is —C═O.
 3. Thecompound or salt of claim 1 wherein each D is O.
 4. The compound

or a pharmaceutically acceptable salt thereof.
 5. A pharmaceuticalcomposition comprising the compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.
 6. Aformulation comprising a compound of claim 1, or pharmaceuticallyacceptable salt thereof, wherein the formulation comprises an excipientchosen from purified water, propylene glycol, PEG 400, glycerin, DMA,ethanol, benzyl alcohol, citric acid/sodium citrate (pH3), citricacid/sodium citrate (pH5), tris(hydroxymethyl)amino methane HCl (pH7.0),0.9% saline, and 1.2% saline, or any combination thereof.
 7. Aformulation comprising a compound of claim 1, or pharmaceuticallyacceptable salt thereof, wherein the formulation comprises an excipientchosen from 20% w/v propylene glycol in saline, 30% w/v propylene glycolin saline, 40% w/v propylene glycol in saline, 50% w/v propylene glycolin saline, 15% w/v propylene glycol in purified water, 30% w/v propyleneglycol in purified water, 50% w/v propylene glycol in purified water,30% w/v propylene glycol and 5 w/v ethanol in purified water, 15% w/vglycerin in purified water, 30% w/v glycerin in purified water, 50% w/vglycerin in purified water, 20% w/v Kleptose in purified water, 40% w/vKleptose in purified water, and 25% w/v Captisol in purified water.
 8. Aformulation comprising

or a pharmaceutically acceptable salt thereof, in 20% w/v Kleptose inpurified water.
 9. A method of preparing the compound of claim 4comprising: a) reacting (R)-(−)-N-Boc-3-pyrrolidinol with a strong baseto form a mixture; further reacting the mixture with2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene to form a compoundhaving Formula II

b) reacting the compound of Formula II with an alcohol and a transitionmetal catalyst in the presence of hydrogen to form a compound of FormulaIII

c) adding the compound of Formula III and pyrimidine-4,6-dicarboxylicacid to a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine andN-methylmorpholine to form a compound of Formula IV

d) reacting the compound of Formula IV with N-Boc- guanidine butyricacid to form a compound of Formula V

e) deprotecting the compound of Formula V to produce the compound ofclaim
 4. 10. A method of preparing the compound of claim 4 comprising:a) deprotonating (R)-3-Hydroxypyrrolidine-1-carboxylic acid tent-butylester, and reacting the resultant compound with2-chloro-1,3-dinitro-5-trifluoromethylbenzene to form(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester; b) reducing(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester in the presence of an alcohol, a transition metalcatalyst, and hydrogen to form(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester; c) coupling(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester with pyrimidine-4,6-dicarboxylic acid in thepresence of 1-[(3-(dimethylamino)-propyl)]-3-ethylcarbodiimidehydrochloride to form pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide};d) reacting pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}with ({[tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)pentanoicacid in the presence of phosphorous oxychloride to formpyrimidine-4,6-dicarboxylic acidbis-{[3-(5-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-pentanoylamino)-2-((R)-1-(tert-butoxycarbonyl)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide};e) deprotecting pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-pentanoylamino)-2-((R)-1-(tert-butoxycarbonyl)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}to form crude pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-guanidino-pentanoylamino)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}tetrahydrochloride;and f) purifying crude pyrimidine-4,6-dicarboxylic acidbis-{[3-(5-guanidino-pentanoylamino)-2-((R)-pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]-amide}tetrahydrochlorideby reverse-phase chromatography.
 11. A method of preparing the compoundof claim 4 comprising: a) deprotonating(R)-3-Hydroxypyrrolidine-1-carboxylic acid tert-butyl ester and furtherreacting the resultant compound with2-chloro-1,3-dinitro-5-trifluoromethylbenzene to form(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester; b) reducing(R)-3-(2,6-dinitro-4-trifluoromethylphenoxy)pyrrolidine-1-carboxylicacid tert-butyl ester in the presence of an alcohol, a transition metalcatalyst, and hydrogen, to form(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester; c) coupling(R)-3-(2,6-diamino-4-trifluoromethylphenoxy)-pyrrolidine-1-carboxylicacid tert-butyl ester with pyrimidine-4,6-dicarboxylic acid in thepresence of 1-[(3-(dimethylamino)-propyl)]-3-ethylcarbodiimidehydrochloride to form pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide};d) reacting pyrimidine-4,6-dicarboxylic acidbis-{[3-amino-2-((R)-1-(tert-butoxycarbonyl)pyrrolidin-3-yloxy)-5-trifluoromethyl-phenyl]amide}with N-Cbz acid in the presence of thionyl chloride; e) reducing theresultant compound of d) in the presence of an alcohol, a transitionmetal catalyst, and hydrogen; f) reacting the resultant compound of e)with di-Boc pyrazole; g) deprotecting the resultant compound of f) toproduce the compound of claim
 4. 12. A method of preparing apharmaceutically acceptable salt of the compound of claim 4 comprising:a) reacting (R)-(−)-N-Boc-3-pyrrolidinol with a strong base to form amixture; further reacting the mixture with2-chloro-5-(trifluoromethyl)-1,3-dinitrobenzene to form a compoundhaving Formula II

b) reacting the compound of Formula II with an alcohol and a transitionmetal catalyst in the presence of hydrogen to form a compound of FormulaIII

c1) adding the compound of Formula III and pyrimidine-4,6-dicarboxylicacid to a mixture of 2-chloro-4,6-dimethoxy-1,3,5-triazine andN-methylmorpholine to form a compound of Formula IV

c2) adding the compound of Formula III and pyrimidine-4,6-dicarboxylicacid to a mixture of 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimidehydrochloride (EDC1) and anhydrous pyridine to form a compound ofFormula IV

d) adding the compound of Formula IV with an N-Cbz acid to a solutioncomprising anhydrous pyridine, dimethylaminopropylamine, and any one ofthionyl chloride, POCl₃, (EtO)₂POCl, or oxalyl chloride to form acompound of Formula Va

e) hydrogenlysis of the Cbz group of the compound of Formula Va toproduce the compound of formula VI

f) protecting the compound of Formula VI to produce the compound offormula VII

g) deprotecting the compound of Formula VII to produce apharmaceutically acceptable salt of the compound of claim 4.